Nucleic Acid Binding Domains and Methods of Use Thereof

ABSTRACT

Provided herein are polypeptides, compositions comprising the polypeptides and methods for genome editing and gene regulation (e.g., activation and/or repression) using the polypeptides or the compositions comprising the polypeptides, such as, DNA binding domains derived from the genus of  Ralstonia . Also disclosed are DNA binding proteins that include a fragment of N-cap sequence of a TALE protein, such as, a  Xanthomonas  TALE protein. Also disclosed are DNA binding proteins that include a fragment of N-cap sequence of a DNA binding protein derived from bacteria of the genus  Ralstonia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority pursuant to 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/690,888, filed Jun. 27, 2018, U.S. Provisional Application No. 62/694,239, filed Jul. 5, 2018, U.S. Provisional Application No. 62/716,147, filed Aug. 8, 2018 and U.S. Provisional Application No. 62/852,134, filed May 23, 2019, the disclosures of which are incorporated herein by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file, “ALTI-718WO Seq List_ST25.txt,” created on Jun. 26, 2019 and having a size of 448 KB. The contents of the text file are incorporated by reference herein in their entirety.

INTRODUCTION

Genome editing and gene regulation techniques include the use of nucleic acid binding domains linked to a functional domain. Provided herein are polypeptides and methods for genome editing and gene regulation, wherein the nucleic acid binding domain is derived from DNA binding proteins from bacteria from the genus of Ralstonia or from Xanthomonas.

SUMMARY

In various aspects, the present disclosure provides a polypeptide comprising a modular nucleic acid binding domain comprising a potency for a target site greater than 65% and a specificity ratio for the target site of at least 50:1; and a functional domain; wherein: the modular nucleic acid binding domain comprises a plurality of repeat units; at least one repeat unit of the plurality of repeat units comprises a binding region configured to bind to a target nucleic acid base within the target site; the potency comprises indel percentage at the target site, and wherein the specificity ratio comprises indel percentage at the target site over indel percentage at a top-ranked off-target site of the polypeptide.

In some aspects, the at least one repeat unit comprises a sequence of A₁₋₁₁X₁X₂B₁₄₋₃₅, wherein: each amino acid residue of A₁₋₁₁ comprises any amino acid residue; X₁X₂ comprises the binding region; each amino acid residue of B₁₄₋₃₅ comprises any amino acid; and a first repeat unit of the plurality of repeat units comprises at least one residue in A₁₋₁₁, B₁₄₋₃₅, or a combination thereof that differs from a corresponding residue in a second repeat unit of the plurality of repeat units.

In various aspects, the present disclosure provides a polypeptide comprising a modular nucleic acid binding domain and a functional domain, wherein: the modular nucleic acid binding domain comprises a plurality of repeat units; at least one repeat unit of the plurality comprises a sequence of A₁₋₁₁X₁X₂B₁₄₋₃₅; each amino acid residue of A₁₋₁₁ comprises any amino acid residue; X₁X₂ comprises a binding region configured to bind to a target nucleic acid base within a target site; each amino acid residue of B₁₄₋₃₅ comprises any amino acid; and a first repeat unit of the plurality of repeat units comprises at least one residue in A₁₋₁₁, B₁₄₋₃₅, or a combination thereof that differs from a corresponding residue in a second repeat unit of the plurality of repeat units.

In some aspects, the binding region comprises an amino acid residue at position 13 or an amino acid residue at position 12 and the amino acid residue at position 13. In further aspects, the amino acid residue at position 13 binds to the target nucleic acid base. In some aspects, the amino acid residue at position 12 stabilizes the configuration of the binding region.

In some aspects, the modular nucleic acid binding domain further comprises a potency for the target site greater than 65% and a specificity ratio for the target site of at least 50:1, wherein the potency comprises indel percentage at the target site and the specificity ratio comprises indel percentage at the target site over indel percentage at a top-ranked off-target site of the polypeptide. In further aspects, the indel percentage is measured by deep sequencing. In some aspects, the modular nucleic acid binding domain further comprises one or more properties selected from the following: (a) binds the target site, wherein the target site comprises a 5′ guanine; (b) comprises from 7 repeat units to 25 repeat units; (c) upon binding to the target site, the modular nucleic acid binding domain is separated from a second modular nucleic acid binding domain bound to a second target site by from 2 to 50 base pairs.

In some aspects, the modular nucleic acid binding domain comprises a Ralstonia repeat unit. In further aspects, the Ralstonia repeat unit is a Ralstonia solanacearum repeat unit. In still further aspects, the B₁₄₋₃₅ of at least one repeat unit of the plurality of repeat units has at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% sequence identity to GGKQALEAVRAQLLDLRAAPYG (SEQ ID NO: 280).

In some aspects, the binding region comprises HD binding to cytosine, NG binding to thymidine, NK binding to guanine, SI binding to adenosine, RS binding to adenosine, HN binding to guanine, or NT binds to adenosine. In some aspects, the at least one repeat unit comprises any one of SEQ ID NO: 267-SEQ ID NO: 279.

In further aspects, the at least one repeat unit comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% sequence identity with any one of SEQ ID NO: 168-SEQ ID NO: 263. In further aspects, the at least one repeat unit comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% sequence identity with SEQ ID NO: 209, SEQ ID NO: 197, SEQ ID NO: 233, SEQ ID NO: 253, SEQ ID NO: 203, or SEQ ID NO: 218. In some aspects, the at least one repeat unit comprises any one of SEQ ID NO: 168-SEQ ID NO: 263. In further aspects, the at least one repeat unit comprises SEQ ID NO: 209, SEQ ID NO: 197, SEQ ID NO: 233, SEQ ID NO: 253, SEQ ID NO: 203, or SEQ ID NO: 218.

In some aspects, the target nucleic acid base is cytosine, guanine, thymidine, adenosine, uracil or a combination thereof. In some aspects, the target site is a nucleic acid sequence within a PDCD1 gene, a CTLA4 gene, a LAG3 gene, a TET2 gene, a BTLA gene, a HAVCR2 gene, a CCR5 gene, a CXCR4 gene, a TRA gene, a TRB gene, a B2M gene, an albumin gene, a HBB gene, a HBA1 gene, a TTR gene, a NR3C1 gene, a CD52 gene, an erythroid specific enhancer of the BCL11A gene, a CBLB gene, a TGFBR1 gene, a SERPINA1 gene, a HBV genomic DNA in infected cells, a CEP290 gene, a DMD gene, a CFTR gene, an IL2RG gene, or a combination thereof.

In other aspects, a nucleic acid sequence encoding a chimeric antigen receptor (CAR), alpha-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), or Factor 9 (F9), is inserted at the target site.

In some aspects, the modular nucleic acid binding domain comprises an N-terminus amino acid sequence, a C-terminus amino acid sequence, or a combination thereof. In further aspects, the N-terminus amino acid sequence is from Xanthomonas spp., Legionella quateirensis, or Ralstonia solanacearum. In still further aspects, the N-terminus amino acid sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% sequence identity to SEQ ID NO: 264, SEQ ID NO: 300, SEQ ID NO: 335, SEQ ID NO: 303, SEQ ID NO: 301, SEQ ID NO: 304, or SEQ ID NO: 320, SEQ ID NO: 321, or SEQ ID NO: 322. In still further aspects, the N-terminus amino acid sequence comprises SEQ ID NO: 264, SEQ ID NO: 300, SEQ ID NO: 335, SEQ ID NO: 303, SEQ ID NO: 301, SEQ ID NO: 304, or SEQ ID NO: 320, SEQ ID NO: 321, or SEQ ID NO: 322.

In some aspects, the C-terminus amino acid sequence is from Xanthomonas spp., Legionella quateirensis, or Ralstonia solanacearum. In further aspects, the C-terminus amino acid sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% sequence identity sequence identity to SEQ ID NO: 266, SEQ ID NO: 298, or SEQ ID NO: 306. In still further aspects, the C-terminus amino acid sequence comprises SEQ ID NO: 266, SEQ ID NO: 298, or SEQ ID NO: 306. In some aspects, the C-terminus amino acid sequence serves as a linker between the modular nucleic acid binding domain and the cleavage domain.

In some aspects, the modular nucleic acid binding domain comprises a half repeat. In further aspects, the half repeat comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% sequence identity sequence identity to SEQ ID NO: 265, SEQ ID NO: 327-SEQ ID NO: 329, or SEQ ID NO: 290. In further aspects, the half repeat comprises SEQ ID NO: 265, SEQ ID NO: 327-SEQ ID NO: 329, or SEQ ID NO: 290.

In still further aspects, the functional domain is a cleavage domain or a repression domain. In some aspects, the cleavage domain comprises at least 33.3% divergence from SEQ ID NO: 163 and is immunologically orthogonal to SEQ ID NO: 163. In further aspects, the polypeptide comprises one or more of the following characteristics: (a) induces greater than 1% indels at a target site; (b) the cleavage domain comprises a molecular weight of less than 23 kDa; (c) the cleavage domain comprises less than 196 amino acids; (d) capable of cleaving across a spacer region greater than 24 base pairs.

In some aspects, the polypeptide induces greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% indels at the target site. In some aspects, the cleavage domain comprises at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% divergence from SEQ ID NO: 163. In some aspects, the cleavage domain comprises a sequence selected from SEQ ID NO: 316-SEQ ID NO: 319.

In further aspects, the cleavage domain comprises a nucleic acid sequence encoding for a sequence having at least 80% sequence identity with SEQ ID NO: 1-SEQ ID NO: 81. In still further aspects, the cleavage domain comprises a nucleic acid sequence encoding for a sequence selected from SEQ ID NO: 1-SEQ ID NO: 81. In some aspects, the nucleic acid sequence comprises at least 80% sequence identity with SEQ ID NO: 82-SEQ ID NO: 162. In further aspects, the nucleotide sequence encoding for the sequence comprises any one of SEQ ID NO: 82-SEQ ID NO: 162.

In some aspects, the repression domain comprises KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, or MeCP2.

In some aspects, the at least one repeat unit comprises 1-20 additional amino acid residues at the C-terminus. In some aspects, the at least repeat unit of the plurality of repeat units is separated from a neighboring repeat unit by a linker. In further aspects, the linker comprises a recognition site. In some aspects, the recognition site is for a small molecule, a protease, or a kinase. In some aspects, the recognition site serves as a localization signal. In some aspects, the plurality of repeat units comprises 3 to 60 repeat units.

In some aspects, a repeat unit of the plurality of repeat units recognizes a target nucleic acid base and wherein the plurality of repeat units has one or more of the following characteristics: (a) at least one repeat unit comprising greater than 39 amino acid residues; (b) at least one repeat unit comprising greater than 35 amino acid residues derived from the genus of Ralstonia; (c) at least one repeat unit comprising less than 32 amino acid residues; and (d) each repeat unit of the plurality of repeat units is separated from a neighboring repeat unit by a linker comprising a recognition site. In some aspects, the at least one repeat unit comprises an amino acid selected from glycine, alanine, threonine or histidine at a position after an amino acid residue at position 35. In some aspects, the at least one repeat unit comprises an amino acid selected from glycine, alanine, threonine or histidine at a position after an amino acid residue at position 39.

Also provided herein is a non-naturally occurring DNA binding polypeptide that includes from N- to C-terminus: a N-terminus region comprising at least residues N+110 to N+1 of a TALE protein, where the N-terminus region does not include residues N+288 to N+116 of the TALE protein; a plurality of TALE repeat units derived from a TALE protein; and C-terminus region of a TALE protein. The N-terminus region may not include at least amino acids N+288 to N+116 of the TALE protein. The N-terminus region may not include amino acids N+288 to up to N+116 of the TALE protein. The N-terminus region may not include at least amino acids N+288 to up to N+111 of the TALE protein. The N-terminus region may include residues N+1 to up to N+115 of the TALE protein. The N-terminus region may include residues N+1 to up to N+110 of the TALE protein. The C-terminus region may include full length C-terminus region of a TALE protein or a fragment thereof, e.g., residues C+1 to C+63 of the TALE protein. The DNA binding polypeptide may be fused to a heterologous functional domain, such as, enzyme, a transcriptional activator, a transcriptional repressor, or a DNA nucleotide modifier. The N-terminus region, the TALE repeat units, and the C-terminus region may be derived from the same TALE protein or from different TALE proteins. The TALE proteins from which the N-terminus region, the TALE repeat units, and the C-terminus region may be derived include Xanthomonas TALE proteins, such as, AvrBs3, AVRHAH1, AvrXa7, AVRB6, or AvrXa10.

In various aspects, the present disclosure provides a method of genome editing, the method comprising: administering any of the above polypeptides or compositions thereof and inducing a double stranded break.

In various aspects, the present disclosure provides method of gene repression, the method comprising administering any of the above polypeptides or compositions thereof and repressing gene expression.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1C show schematics of the domain structure of DNA binding proteins (not drawn to scale).

FIG. 2 shows nuclease activity mediated by DNA binding protein dimers that each include from N-terminus to C-terminus: a N-terminus region of a TALE protein, TALE repeat units, C-terminus region of a TALE protein, and a Fok1 endonuclease.

DETAILED DESCRIPTION

The present disclosure provides modular nucleic acid binding domains (NBDs) derived from the genus of bacteria. For example, in some embodiments, the present disclosure provides NBDs derived from bacteria that serve as plant pathogens, such as from the genus of Xanthomonas spp. and Ralstonia. In particular embodiments, the present disclosure provides NBDs from the genus of Ralstonia. Also provided herein are NBDs from the animal pathogen, Legionella. Provided herein are sequences of repeat units derived from the genus of Ralstonia, which can be linked together to form non-naturally occurring modular nucleic acid binding domains (NBDs), capable of targeting and binding any target nucleic acid sequence (e.g., DNA sequence).

In some embodiments, “derived” indicates that a protein is from a particular source (e.g., Ralstonia), is a variant of a protein from a particular source (e.g., Ralstonia), is a mutated or modified form of the protein from a particular source (e.g., Ralstonia), and shares at least 30% sequence identity with, at least 40% sequence identity with, at least 50% sequence identity with, at least 60% sequence identity with, at least 70% sequence identity with, at least 80% sequence identity with, or at least 90% sequence identity with a protein from a particular source (e.g., Ralstonia).

In some embodiments, “modular” indicates that a particular polypeptide such as a nucleic acid binding domain, comprises a plurality of repeat units that can be switched and replaced with other repeat units. For example, any repeat unit in a modular nucleic acid binding domain can be switched with a different repeat unit. In some embodiments, modularity of the nucleic acid binding domains disclosed herein allows for switching the target nucleic acid base for a particular repeat unit by simply switching it out for another repeat unit. In some embodiments, modularity of the nucleic acid binding domains disclosed herein allows for swapping out a particular repeat unit for another repeat unit to increase the affinity of the repeat unit for a particular target nucleic acid. Overall, the modular nature of the nucleic acid binding domains disclosed herein enables the development of genome editing complexes that can precisely target any nucleic acid sequence of interest.

In particular embodiments, modular nucleic acid binding domains (NBDs), also referred to herein as “DNA binding polypeptides,” are provided herein from the genus of Ralstonia solanacearum. In some embodiments, modular nucleic acid binding domains derived from Ralstonia (RNBDs) can be engineered to bind to a target gene of interest for purposes of gene editing or gene regulation. An RNBD can be engineered to target and bind a specific nucleic acid sequence. The nucleic acid sequence can be DNA or RNA.

In some embodiments, the RNBD can comprise a plurality of repeat units, wherein each repeat unit recognizes and binds to a single nucleotide (in DNA or RNA) or base pair. Each repeat unit in the plurality of repeat units can be specifically selected to target and bind to a specific nucleic acid sequence, thus contributing to the modular nature of the DNA binding polypeptide. A non-naturally occurring Ralstonia-derived modular nucleic acid binding domain can comprise a plurality of repeat units, wherein each repeat unit of the plurality of repeat units recognizes a single target nucleotide, base pair, or both.

Ralstonia-Derived DNA Binding Domains

In some embodiments, the repeat unit of a modular nucleic acid binding domain can be derived from a bacterial protein. For example, the bacterial protein can be a transcription activator like effector-like protein (TALE-like protein). The bacterial protein can be derived from Ralstonia solanacearum. Repeat units derived from Ralstonia solanacearum can be 33-35 amino acid residues in length. In some embodiments, the repeat unit can be derived from the naturally occurring Ralstonia solanacearum TALE-like protein.

TABLE 1 below shows exemplary repeat units derived from the genus of Ralstonia, which are capable of binding a target nucleic acid.

TABLE 1 Exemplary Ralstonia-derived Repeat Units SEQ ID NO Sequence SEQ ID LDTEQVVAIASHNGGKQALEAVKADLLDLLGAPYV NO: 168 SEQ ID LDTEQVVAIASHNGGKQALEAVKADLLDLRGAPYA NO: 169 SEQ ID LDTEQVVAIASHNGGKQALEAVKADLLELRGAPYA NO: 170 SEQ ID LDTEQVVAIASHNGGKQALEAVKAHLLDLRGAPYA NO: 171 SEQ ID LNTEQVVAIASHNGGKQALEAVKADLLDLRGAPYA NO: 172 SEQ ID LNTEQVVAIASNNGGKQALEAVKTHLLDLRGARYA NO: 173 SEQ ID LNTEQVVAIASNPGGKQALEAVRALFPDLRAAPYA NO: 174 SEQ ID LNTEQVVAIASSHGGKQALEAVRALFPDLRAAPYA NO: 175 SEQ ID LNTEQVVAVASNKGGKQALEAVGAQLLALRAVPYA NO: 176 SEQ ID LNTEQVVAVASNKGGKQALEAVGAQLLALRAVPYE NO: 177 SEQ ID LSAAQVVAIASHDGGKQALEAVGTQLVALRAAPYA NO: 178 SEQ ID LSIAQVVAVASRSGGKQALEAVRAQLLALRAAPYG NO: 179 SEQ ID LSPEQVVAIASNHGGKQALEAVRALFRGLRAAPYG NO: 180 SEQ ID LSPEQVVAIASNNGGKQALEAVKAQLLELRAAPYE NO: 181 SEQ ID LSTAQLVAIASNPGGKQALEAIRALFRELRAAPYA NO: 182 SEQ ID LSTAQLVAIASNPGGKQALEAVRALFRELRAAPYA NO: 183 SEQ ID LSTAQLVAIASNPGGKQALEAVRAPFREVRAAPYA NO: 184 SEQ ID LSTAQLVSIASNPGGKQALEAVRALFRELRAAPYA NO: 185 SEQ ID LSTAQVAAIASHDGGKQALEAVGTQLVVLRAAPYA NO: 186 SEQ ID LSTAQVATIASSIGGRQALEALKVQLPVLRAAPYG NO: 187 SEQ ID LSTAQVATIASSIGGRQALEAVKVQLPVLRAAPYG NO: 188 SEQ ID LSTAQVVAIAANNGGKQALEAVRALLPVLRVAPYE NO: 189 SEQ ID LSTAQVVAIAGNGGGKQALEGIGEQLLKLRTAPYG NO: 190 SEQ ID LSTAQVVAIASHDGGKQALEAAGTQLVALRAAPYA NO: 191 SEQ ID LSTAQVVAIASHDGGKQALEAVGAQLVELRAAPYA NO: 192 SEQ ID LSTAQVVAIASHDGGKQALEAVGTQLVALRAAPYA NO: 193 SEQ ID LSTAQVVAIASHDGGNQALEAVGTQLVALRAAPYA NO: 194 SEQ ID LSTAQVVAIASHNGGKQALEAVKAQLLDLRGAPYA NO: 195 SEQ ID LSTAQVVAIASNDGGKQALEEVEAQLLALRAAPYE NO: 196 SEQ ID LSTAQVVAIASNGGGKQALEGIGEQLLKLRTAPYG NO: 197 SEQ ID LSTAQVVAIASNGGGKQALEGIGEQLRKLRTAPYG NO: 198 SEQ ID LSTAQVVAIASNPGGKQALEAVRALFRELRAAPYA NO: 199 SEQ ID LSTAQVVAIASQNGGKQALEAVKAQLLDLRGAPYA NO: 200 SEQ ID LSTAQVVAIASSHGGKQALEAVRALFRELRAAPYG NO: 201 SEQ ID LSTAQVVAIASSNGGKQALEAVWALLPVLRATPYD NO: 202 SEQ ID LSTAQVVAIATRSGGKQALEAVRAQLLDLRAAPYG NO: 203 SEQ ID LSTAQVVAVAGRNGGKQALEAVRAQLPALRAAPYG NO: 204 SEQ ID LSTAQVVAVASSNGGKQALEAVWALLPVLRATPYD NO: 205 SEQ ID LSTAQVVTIASSNGGKQALEAVWALLPVLRATPYD NO: 206 SEQ ID LSTEQVVAIAGHDGGKQALEAVGAQLVALRAAPYA NO: 207 SEQ ID LSTEQVVAIASHDGGKQALEAVGAQLVALLAAPYA NO: 208 SEQ ID LSTEQVVAIASHDGGKQALEAVGAQLVALRAAPYA NO: 209 SEQ ID LSTEQVVAIASHDGGKQALEAVGGQLVALRAAPYA NO: 210 SEQ ID LSTEQVVAIASHDGGKQALEAVGTQLVALRAAPYA NO: 211 SEQ ID LSTEQVVAIASHDGGKQALEAVGVQLVALRAAPYA NO: 212 SEQ ID LSTEQVVAIASHDGGKQALEAVVAQLVALRAAPYA NO: 213 SEQ ID LSTEQVVAIASHDGGKQPLEAVGAQLVALRAAPYA NO: 214 SEQ ID LSTEQVVAIASHGGGKQVLEGIGEQLLKLRAAPYG NO: 215 SEQ ID LSTEQVVAIASHKGGKQALEGIGEQLLKLRAAPYG NO: 216 SEQ ID LSTEQVVAIASHNGGKQALEAVKADLLDLRGAPYA NO: 217 SEQ ID LSTEQVVAIASHNGGKQALEAVKADLLELRGAPYA NO: 218 SEQ ID LSTEQVVAIASHNGGKQALEAVKAHLLDLRGAPYA NO: 219 SEQ ID LSTEQVVAIASHNGGKQALEAVKAHLLDLRGVPYA NO: 220 SEQ ID LSTEQVVAIASHNGGKQALEAVKAHLLELRGAPYA NO: 221 SEQ ID LSTEQVVAIASHNGGKQALEAVKAQLLDLRGAPYA NO: 222 SEQ ID LSTEQVVAIASHNGGKQALEAVKAQLLELRGAPYA NO: 223 SEQ ID LSTEQVVAIASHNGGKQALEAVKAQLPVLRRAPYG NO: 224 SEQ ID LSTEQVVAIASHNGGKQALEAVKTQLLELRGAPYA NO: 225 SEQ ID LSTEQVVAIASHNGGKQALEAVRAQLPALRAAPYG NO: 226 SEQ ID LSTEQVVAIASHNGSKQALEAVKAQLLDLRGAPYA NO: 227 SEQ ID LSTEQVVAIASNGGGKQALEGIGKQLQELRAAPHG NO: 228 SEQ ID LSTEQVVAIASNGGGKQALEGIGKQLQELRAAPYG NO: 229 SEQ ID LSTEQVVAIASNHGGKQALEAVRALFRELRAAPYA NO: 230 SEQ ID LSTEQVVAIASNHGGKQALEAVRALFRGLRAAPYG NO: 231 SEQ ID LSTEQVVAIASNKGGKQALEAVKADLLDLRGAPYV NO: 232 SEQ ID LSTEQVVAIASNKGGKQALEAVKAHLLDLLGAPYV NO: 233 SEQ ID LSTEQVVAIASNKGGKQALEAVKAQLLALRAAPYA NO: 234 SEQ ID LSTEQVVAIASNKGGKQALEAVKAQLLELRGAPYA NO: 235 SEQ ID LSTEQVVAIASNNGGKQALEAVKALLLELRAAPYE NO: 236 SEQ ID LSTEQVVAIASNNGGKQALEAVKAQLLALRAAPYE NO: 237 SEQ ID LSTEQVVAIASNNGGKQALEAVKAQLLDLRGAPYA NO: 238 SEQ ID LSTEQVVAIASNNGGKQALEAVKAQLLVLRAAPYG NO: 239 SEQ ID LSTEQVVAIASNNGGKQALEAVKAQLPALRAAPYE NO: 240 SEQ ID LSTEQVVAIASNNGGKQALEAVKAQLPVLRRAPCG NO: 241 SEQ ID LSTEQVVAIASNNGGKQALEAVKAQLPVLRRAPYG NO: 242 SEQ ID LSTEQVVAIASNNGGKQALEAVKARLLDLRGAPYA NO: 243 SEQ ID LSTEQVVAIASNNGGKQALEAVKTQLLALRTAPYE NO: 244 SEQ ID LSTEQVVAIASNPGGKQALEAVRALFPDLRAAPYA NO: 245 SEQ ID LSTEQVVAIASSHGGKQALEAVRALFPDLRAAPYA NO: 246 SEQ ID LSTEQVVAIASSHGGKQALEAVRALLPVLRATPYD NO: 247 SEQ ID LSTEQVVAVASHNGGKQALEAVRAQLLDLRAAPYE NO: 248 SEQ ID LSTEQVVAVASNKGGKQALAAVEAQLLRLRAAPYE NO: 249 SEQ ID LSTEQVVAVASNKGGKQALEEVEAQLLRLRAAPYE NO: 250 SEQ ID LSTEQVVAVASNKGGKQVLEAVGAQLLALRAVPYE NO: 251 SEQ ID LSTEQVVAVASNNGGKQALKAVKAQLLALRAAPYE NO: 252 SEQ ID LSTEQVVVIANSIGGKQALEAVKVQLPVLRAAPYE NO: 253 SEQ ID LSTGQVVAIASNGGGRQALEAVREQLLALRAVPYE NO: 254 SEQ ID LSPEQVVTIASNNGGKQALEAVRAQLLALRAAPYG NO: 255 SEQ ID LTIAQVVAVASHNGGKQALEAIGAQLLALRAAPYA NO: 256 SEQ ID LTIAQVVAVASHNGGKQALEVIGAQLLALRAAPYA NO: 257 SEQ ID LTPQQVVAIAANTGGKQALGAITTQLPILRAAPYE NO: 258 SEQ ID LTPQQVVAIASNTGGKQALEAVTVQLRVLRGARYG NO: 259 SEQ ID LTPQQVVAIASNTGGKRALEAVCVQLPVLRAAPYR NO: 260 SEQ ID LTPQQVVAIASNTGGKRALEAVRVQLPVLRAAPYE NO: 261 SEQ ID LTTAQVVAIASNDGGKQALEAVGAQLLVLRAVPYE NO: 262 SEQ ID LTTAQVVAIASNDGGKQTLEVAGAQLLALRAVPYE NO: 263 SEQ ID LSTAQVVAVASGSGGKPALEAVRAQLLALRAAPYG NO: 336 SEQ ID LSTAQVVAVASGSGGKPALEAVRAQLLALRAAPYG NO: 337 SEQ ID LNTAQIVAIASHDGGKPALEAVWAKLPVLRGAPYA NO: 338 SEQ ID LNTAQVVAIASHDGGKPALEAVRAKLPVLRGVPYA NO: 339 SEQ ID LNTAQVVAIASHDGGKPALEAVWAKLPVLRGVPYA NO: 340 SEQ ID LNTAQVVAIASHDGGKPALEAVWAKLPVLRGVPYE NO: 341 SEQ ID LSTAQVVAIASHDGGKPALEAVWAKLPVLRGAPYA NO: 342 SEQ ID LSTAQVVAVASHDGGKPALEAVRKQLPVLRGVPHQ NO: 343 SEQ ID LSTAQVVAVASHDGGKPALEAVRKQLPVLRGVPHQ NO: 344 SEQ ID LNTAQVVAIASHDGGKPALEAVWAKLPVLRGVPYA NO: 345 SEQ ID LSTEQVVAIASHNGGKLALEAVKAHLLDLRGAPYA NO: 346 SEQ ID LSTEQVVAIASHNGGKPALEAVKAHLLALRAAPYA NO: 347 SEQ ID LNTAQVVAIASHYGGKPALEAVWAKLPVLRGVPYA NO: 348 SEQ ID LNTEQVVAIASNNGGKPALEAVKAQLLELRAAPYE NO: 349 SEQ ID LSPEQVVAIASNNGGKPALEAVKALLLALRAAPYE NO: 350 SEQ ID LSPEQVVAIASNNGGKPALEAVKAQLLELRAAPYE NO: 351 SEQ ID LSTEQVVAIASNNGGKPALEAVKALLLALRAAPYE NO: 352 SEQ ID LSTEQVVAIASNNGGKPALEAVKALLLELRAAPYE NO: 353 SEQ ID LSPEQVVAIASNNGGKPALEAVKALLLALRAAPYE NO: 354 SEQ ID LSPEQVVAIASNNGGKPALEAVKAQLLELRAAPYE NO: 355 SEQ ID LSTEQVVAIASNNGGKPALEAVKALLLELRAAPYE NO: 356

In some embodiments, an RNBD of the present disclosure can comprise between 1 to 50 Ralstonia solanacearum-derived repeat units. In some embodiments, an RNBD of the present disclosure can comprise between 9 and 36 Ralstonia solanacearum-derived repeat units. Preferably, in some embodiments, an RNBD of the present disclosure can comprise between 12 and 30 Ralstonia solanacearum-derived repeat units. A RNBD described herein can comprise between 5 to 10 Ralstonia solanacearum-derived repeat units, between 10 to 15 Ralstonia solanacearum-derived repeat units, between 15 to 20 Ralstonia solanacearum-derived repeat units, between 20 to 25 Ralstonia solanacearum-derived repeat units, between 25 to 30 Ralstonia solanacearum-derived repeat units, or between 30 to 35 Ralstonia solanacearum-derived repeat units, between 35 to 40 Ralstonia solanacearum-derived repeat units. A RNBD described herein can comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or more Ralstonia solanacearum-derived repeat units.

A Ralstonia solanacearum-derived repeat unit can be derived from a wild-type repeat unit, such as any one of SEQ ID NO: 168-SEQ ID NO: 263 or SEQ ID NO: 336-SEQ ID NO: 356. A Ralstonia solanacearum-repeat unit can have at least 80% sequence identity with any one of SEQ ID NO: 168-SEQ ID NO: 263 or SEQ ID NO: 336-SEQ ID NO: 356. A Ralstonia solanacearum-derived repeat unit can also comprise a modified Ralstonia solanacearum-derived repeat unit enhanced for specific recognition of a nucleotide or base pair. An RNBD described herein can comprise one or more wild-type Ralstonia solanacearum-derived repeat units, one or more modified Ralstonia solanacearum-derived repeat units, or a combination thereof. In some embodiments, a modified Ralstonia solanacearum-derived repeat unit can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 mutations that can enhance recognition of a specific nucleotide or base pair. In some embodiments, a modified Ralstonia solanacearum-derived repeat unit can comprise more than 1 modification, for example 1 to 5 modifications, 5 to 10 modifications, 10 to 15 modifications, 15 to 20 modifications, 20 to 25 modification, or 25-29 modifications. In some embodiments, An RNBD can comprise more than one modified Ralstonia solanacearum-derived repeat units, wherein each of the modified Ralstonia solanacearum-derived repeat units can have a different number of modifications.

The Ralstonia solanacearum-derived repeat units comprise amino acid residues at positions 12 and 13, what is referred to herein as, a repeat variable diresidue (RVD). The RVD can modulate binding affinity of the repeat unit for a particular nucleic acid base (e.g., adenosine, guanine, cytosine, thymidine, or uracil (in RNA sequences)). In some embodiments, a single amino acid residue can modulate binding to the target nucleic acid base. In some embodiments, two amino acid residues (RVD) can modulate binding to the target nucleic acid base. In some embodiments, any repeat unit disclosed herein can have an RVD selected from HD, HG, HK, HN, ND, NG, NH, NK, NN, NP, NT, QN, RN, RS, SH, SI, or SN. In some embodiments, an RVD of HD can bind to cytosine. In some embodiments, an RVD of NG can bind to thymidine. In some embodiments, an RVD of NK can bind to guanine. In some embodiments, an RVD of SI can bind to adenosine. In some embodiments, an RVD of RS can bind to adenosine. In some embodiments, an RVD of HN can bind to guanine. In some embodiments, an RVD of NT can bind to adenosine.

In some embodiments, a repeat unit having at least 80% sequence identity with SEQ ID NO: 209 can be included in a DNA binding domain of the present disclosure to bind to cytosine. In some embodiments, a repeat unit having at least 80% sequence identity with SEQ ID NO: 197 can be included in a DNA binding domain of the present disclosure to bind to thymidine. In some embodiments, a repeat unit having at least 80% sequence identity with SEQ ID NO: 233 can be included in a DNA binding domain of the present disclosure to bind to guanine. In some embodiments, a repeat unit having at least 80% sequence identity with SEQ ID NO: 253 can be included in a DNA binding domain of the present disclosure to bind to adenosine. In some embodiments, a repeat unit having at least 80% sequence identity with SEQ ID NO: 203 can be included in a DNA binding domain of the present disclosure to bind to adenosine. In some embodiments, a repeat unit having at least 80% sequence identity with SEQ ID NO: 218 can be included in a DNA binding domain of the present disclosure to bind to guanine. In some embodiments, the repeat unit of SEQ ID NO: 209 can be included in a DNA binding domain of the present disclosure to bind to cytosine. In some embodiments, the repeat unit of SEQ ID NO: 197 can be included in a DNA binding domain of the present disclosure to bind to thymidine. In some embodiments, the repeat unit of SEQ ID NO: 233 can be included in a DNA binding domain of the present disclosure to bind to guanine. In some embodiments, the repeat unit of SEQ ID NO: 253 can be included in a DNA binding domain of the present disclosure to bind to adenosine. In some embodiments, the repeat unit of SEQ ID NO: 203 can be included in a DNA binding domain of the present disclosure to bind to adenosine. In some embodiments, the repeat unit of SEQ ID NO: 218 can be included in a DNA binding domain of the present disclosure to bind to guanine.

In some embodiments, the present disclosure provides repeat units as set forth in SEQ ID NO: 267-SEQ ID NO: SEQ ID NO: 279. Unspecified amino acid residues in SEQ ID NO: 267-SEQ ID NO: SEQ ID NO: 279 can be any amino acid residues. In particular embodiments, unspecified amino acid residues in SEQ ID NO: 267-SEQ ID NO: SEQ ID NO: 279 can be those set forth in the Variable Definition column of TABLE 2.

TABLE 2 shows consensus sequences of Ralstonia-derived repeat units.

TABLE 2 Consensus Sequences of Ralstonia-derived Repeat Units RVD Consensus Sequence Variable Definition HN LX₁X₂X₃QVVX₄X₅ASHNGX₆ X₁: D|N|S|T, X₂: I|T|V, X₃: A|E, KQALEX₇X₈X9X₁₀X₁₁LX₁₂ X₄: A|T, X₅: I|V, X₆: G|S, X₇: X₁₃LX₁₄X₁₅X₁₆PYX₁₇ (SEQ A|V, X₈: I|V, X₉: G|K|R, X₁₀: ID NO: 267) A|T, X₁₁: D|H|Q, X₁₂: L|P, X₁₃: A|D|E|V, X₁₄: L|R, X₁₅: A|G|R, X16: A|V, X₁₇: A|E|G|V NN LX₁X₂X₃QVVAX₄AX₅NNGG X₁: N|S, X₂: P|T, X₃: A|E, X₄: KQALX₆AVX₇X₈X₉LX₁₀X₁₁ I|V, X₅: A|S, X₆: E|K, X₇: K|R, LRX₁₂AX₁₃X₁₄X₁₅ (SEQ ID X₈: A|T, X₉: H|L|Q|R, X₁₀: L|P, NO: 268) X₁₁: A|D|E|V, X₁₂: A|G|R|T|V, X₁₃: P|R, X₁₄: C|Y, X₁₅: A|E|G NP LX₁TX₂QX₃VX₄IASNPGGK X₁: N|S, X₂: A|E, X₃: L|V, X₄: QALEAX₅RAX₆FX₇X₈X₉RA A|S, X₅: I|V, X₆: L|P, X₇: P|R, APYA (SEQ ID NO: 269) X₈: D|E, X₉: L|V SH LX₁TX₂QVVAIASSHGGKQ X₁: N|S, X₂: A|E, X₃: F|L, X₄: ALEAVRALX₃X₄X₅LRAX₆P P|R, X₅: D|E|V, X₆: A|T, X₇: YX₇ (SEQ ID NO: 270) A|D|G NK LX₁TEQVVAX₂ASNKGGKQ X₁: N|S, X₁₀: A|G, X₁₁: A|V, X₃LX₄X₅VX₆AX₇LLX₈LX₉X₁₀ X₁₂: A|E|V, X₂: I|V, X₃: A|V, X₁₁PYX₁₂(SEQ ID NO: 271) X₄: A|E, X₅: A|E, X₆: E|G|K, X₇: D|H|Q, X₈: A|D|E|R, X₉: L|R HD LSX₁X₂QVX₃AIAX₄HDGGX₅ X₁: A|T, X₂: A|E, X₃: A|V, QX₆LEAX₇X₈X₉QLVX₁₀LX₁₁ X₄: G|S, X₅: K|N, X₆: A|P, AAPYA (SEQ ID NO: 272) X₇: A|V, X₈: G|V, X₉: A|G|T|V, X₁₀: A|E|V, X₁₁: L|R RS LSX₁AQVVAX₂AX₃RSGGK X₁: I|T, X₂: I|V, X₃: S|T, X₄: QALEAVRAQLLX₄LRAAP A|D YG (SEQ ID NO: 273) NH LSX₁EQVVAIASNHGGKQ X₁: P|T, X₂: E|G, X₃: A|G ALEAVRALFRX₂LRAAPY X (SEQ ID NO: 274) SI LSTX₁QVX₂X₃IAX₄SIGGX₅ X₁: A|E, X₂: A|V, X₃: T|V, QALEAX₆KVQLPVLRAAP X₄: N|S, X₅: K|R, X₆: L|V, X₇: YX₇ (SEQ ID NO: 275) E|G ND LX₁TAQVVAIASNDGGKQ X₁: S|T, X₂: A|T, X₃: A|E|V, X₂LEX₃X₄X₅AQLLX₆LRAX₇ X₄: A|V, X₅: E|G, X₆: A|V, X₇: PYE (SEQ ID NO: 276) A|V SN LSTAQVVX₁X₂ASSNGGK X₁: A|T, X₂: I|V QALEAVWALLPVLRATP YD (SEQ ID NO: 277) NG LSTX₁QVVAIAX₂NGGGX₃ X₁: A|E|G, X₂: G|S, X₃: K|R, QALEX₄X₅X₆X₇QLX₈X₉LR X₄: A|G, X₅: I|V, X₆: G|R, X₇: X₁₀X₁₁PX₁₂X₁₃ (SEQ ID NO: E|K, X₈: L|Q|R, X₉: A|E|K, X₁₀: 278) A|T, X₁₁: A|V, X₁₂: H|Y, X₁₃: E|G NT LTPQQVVAIAX₁NTGGKX₂ X₁: A|S, X₁₀: P|R, X₁₁: E|G|R, ALX₃AX₄X₅X₆QLX₇X₈LRX₉ X₂: Q|R, X₃: E|G, X₄: I|V, X₅: AX₁₀YX₁₁ (SEQ ID NO: 279) C|R|T, X₆: T|V, X₇: P|R, X₈: I|V, X₉: A|G

In some aspects, the at least one repeat unit comprises any one of SEQ ID NO: 267-SEQ ID NO: 279. In some embodiments, the present disclosure provides a modular nucleic acid binding domain (e.g., RNBD or MAP-NBD), wherein the modular nucleic acid binding domain comprises a repeat unit with a sequence of A₁₋₁₁X₁X₂B₁₄₋₃₅, wherein A₁₋₁₁ comprises 11 amino acid residues and wherein each amino acid residue of A₁₋₁₁ can be any amino acid. In some embodiments, A₁₋₁₁ can be any amino acids in position 1 through position 11 of any one of SEQ ID NO: 168-SEQ ID NO: 263 or SEQ ID NO: 336-SEQ ID NO: 356. X₁X₂ comprises any repeat variable diresidue (RVD) disclosed herein and comprises at least one amino acid at position 12 or position 13. As described herein, this RVD contacts and binds to a target nucleic acid base of a target site. Said RVD can be the RVD of any repeat unit disclosed herein, such as position 12 and position 13 of any one of SEQ ID NO: 168-SEQ ID NO: 263 or SEQ ID NO: 336-SEQ ID NO: 356. B₁₄₋₃₅ can comprise 22 amino acid residues and each amino acid residue of B₁₄₋₃₅ can be any amino acid. In some embodiments, B₁₄₋₃₅ can be any amino acid in position 14 through position 35 of any one of SEQ ID NO: 168-SEQ ID NO: 263 or SEQ ID NO: 336-SEQ ID NO: 356. In particular embodiments, a modular nucleic acid binding domain (e.g., RNBD or MAP-NBD) having the above sequence of A₁₋₁₁X₁X₂B₁₄₋₃₅ can have a first repeat unit with at least one residue in A₁₋₁₁, B₁₄₋₃₅, or a combination thereof that differs from a corresponding residue in a second repeat unit in the modular nucleic acid binding domain (e.g., RNBD or MAP-NBD). In other words, at least two repeat units in a modular nucleic acid binding domain (e.g., RNBD or MAP-NBD) described herein can have different amino acid residues with respect to each other, at the same position outside the RVD region. Thus, in some embodiments, a modular nucleic acid binding domain (e.g., RNBD or MAP-NBD) described herein can have variant backbones with respect to each repeat unit in the plurality of repeat units that make up the modular nucleic acid binding domain. In some embodiments, an RNBD of the present disclosure can have a sequence of GGKQALEAVRAQLLDLRAAPYG (SEQ ID NO: 280) at B14-35.

In some embodiments, the present disclosure provides a polypeptide comprising a modular nucleic acid binding domain and a functional domain, wherein: the modular nucleic acid binding domain comprises a plurality of repeat units; at least one repeat unit of the plurality comprises a sequence of A₁₋₁₁X₁X₂B₁₄₋₃₅; each amino acid residue of A₁₋₁₁ comprises any amino acid residue; X₁X₂ comprises a binding region configured to bind to a target nucleic acid base within a target site; each amino acid residue of B₁₄₋₃₅ comprises any amino acid; and a first repeat unit of the plurality of repeat units comprises at least one residue in A₁₋₁₁, B₁₄₋₃₅, or a combination thereof that differs from a corresponding residue in a second repeat unit of the plurality of repeat units. In some embodiments, the binding region comprises an amino acid residue at position 13 or an amino acid residue at position 12 and the amino acid residue at position 13. In further aspects, the amino acid residue at position 13 binds to the target nucleic acid base. In some aspects, the amino acid residue at position 12 stabilizes the configuration of the binding region.

In some embodiments, the modular nucleic acid binding domain comprises a Ralstonia repeat unit. In further aspects, the Ralstonia repeat unit is a Ralstonia solanacearum repeat unit. In still further aspects, the B₁₄₋₃₅ of at least one repeat unit of the plurality of repeat units has at least 92% sequence identity to GGKQALEAVRAQLLDLRAAPYG (SEQ ID NO: 280).

In some embodiments, a modular nucleic acid binding sequence (e.g., RNBD) can comprise one or more of the following characteristics: the modular nucleic acid binding sequence (e.g., RNBD) can bind a nucleic acid sequence, wherein the target site comprises a 5′ guanine, the modular nucleic acid binding domain (e.g., RNBD) can comprise 7 repeat units to 25 repeat units, a first modular nucleic acid binding sequence (e.g., RNBD) can bind a target nucleic acid sequence and be separated from a second modular nucleic acid binding domain (e.g., RNBD) from 2 to 50 base pairs, or any combination thereof.

In some embodiments, an RNBD of the present disclosure can have the full length naturally occurring N-terminus of a naturally occurring Ralstonia solanacearum-derived protein. In some embodiments, any truncation of the full length naturally occurring N-terminus of a naturally occurring Ralstonia solanacearum-derived protein can be used at the N-terminus of an RNBD of the present disclosure. For example, in some embodiments, amino acid residues at positions 1 (H) to position 137 (F) of the naturally occurring Ralstonia solanacearum-derived protein N-terminus can be used. In particular embodiments, said truncated N-terminus from position 1 (H) to position 137 (F) can have a sequence as follows: FGKLVALGYSREQIRKLKQESLSEIAKYHTTLTGQGFTHADICRISRRRQSLRVVARNYPELA AALPELTRAHIVDIARQRSGDLALQALLPVATALTAAPLRLSASQIATVAQYGERPAIQALY RLRRKLTRAPLH (SEQ ID NO: 264). In some embodiments, the naturally occurring N-terminus of Ralstonia solanacearum can be truncated to any length and used at the N-terminus of the engineered DNA binding domain. For example, the naturally occurring N-terminus of Ralstonia solanacearum can be truncated to amino acid residues at position 1 (H) to position 120 (K) as follows: KQESLSEIAKYHTTLTGQGFTHADICRISRRRQSLRVVARNYPELAAALPELTRAHIVDIARQ RSGDLALQALLPVATALTAAPLRLSASQIATVAQYGERPAIQALYRLRRKLTRAPLH (SEQ ID NO: 303) and used at the N-terminus of the RNBD. The naturally occurring N-terminus of Ralstonia solanacearum can be truncated such that it includes amino acid residues at positions 1 to 115 and used as the N-terminus of the engineered DNA binding domain. In certain aspects, the truncated N-terminus sequence may be at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to the amino acid sequence set forth in SEQ ID NO: 320. The naturally occurring N-terminus of Ralstonia solanacearum can be truncated to amino acid residues at positions 1 to 50, 1 to 70, 1 to 100, 1 to 120, 1 to 130, 10 to 40, 60 to 100, or 100 to 120 and used at the N-terminus of the engineered DNA binding domain. Truncation of the N-termini can be particularly advantageous for obtaining DNA binding domains, which are smaller in size including number of amino acids and overall molecular weight. A reduced number of amino acids can allow for more efficient packaging into a viral vector and a smaller molecular weight can result in more efficient loading of the DNA binding domains in non-viral vectors for delivery.

In some embodiments, the N-terminus, referred to as the amino terminus or the “NH2” domain, can recognize a guanine. In some embodiments, the N-terminus can be engineered to bind a cytosine, adenosine, thymidine, guanine, or uracil.

In some embodiments, an RNBD of the present disclosure can have a DNA binding domain, in which the final full length repeat unit of 33-35 amino acid residues is followed by a half repeat also derived from Ralstonia solanacearum. The half repeat can have 15 to 23 amino acid residues, for example, the half repeat can have 19 amino acid residues. In particular embodiments, the half repeat can have a sequence as follows: LSTAQVVAIACISGQQALE (SEQ ID NO: 265).

In some embodiments, an RNBD of the present disclosure can have the full length naturally occurring C-terminus of a naturally occurring Ralstonia solanacearum-derived protein. In some embodiments, any truncation of the full length naturally occurring C-terminus of a naturally occurring Ralstonia solanacearum-derived protein can be used at the C-terminus of an RNBD of the present disclosure. For example, in some embodiments, the RNBD can comprise amino acid residues at position 1 (A) to position 63 (S) as follows: AIEAHMPTLRQASHSLSPERVAAIACIGGRSAVEAVRQGLPVKAIRRIRREKAPVAGPPPAS (SEQ ID NO: 266) of the naturally occurring Ralstonia solanacearum-derived protein C-terminus. In some embodiments, the naturally occurring C-terminus of Ralstonia solanacearum can be truncated to any length and used at the C-terminus of the RNBD. For example, the naturally occurring C-terminus of Ralstonia solanacearum can be truncated to amino acid residues at positions 1 to 63 and used at the C-terminus of the RNBD. The naturally occurring C-terminus of Ralstonia solanacearum can be truncated amino acid residues at positions 1 to 50 and used at the C-terminus of the RNBD. The naturally occurring C-terminus of Ralstonia solanacearum can be truncated to amino acid residues at positions 1 to 63, 1 to 50, 1 to 70, 1 to 100, 1 to 120, 1 to 130, 10 to 40, 60 to 100, or 100 to 120 and used at the C-terminus of the RNBD.

TABLE 3 shows N-termini, C-termini, and half repeats derived from Ralstonia.

TABLE 3 Ralstonia-Derived N-terminus, C-terminus, and Half-Repeat SEQ ID NO Description Sequence SEQ ID NO: 320 Truncated N-terminus; positions 1 SEIAKYHTTLTGQGFTHADICRISRRRQS (H) to 115 (S) of the naturally LRVVARNYPELAAALPELTRAHIVDIAR occurring Ralstonia solanacearum- QRSGDLALQALLPVATALTAAPLRLSAS derived protein N-terminus QIATVAQYGERPAIQALYRLRRKLTRAP LH SEQ ID NO: 264 Truncated N-terminus; positions 1 FGKLVALGYSREQIRKLKQESLSEIAKYH (H) to 137 (F) of the naturally TTLTGQGFTHADICRISRRRQSLRVVARN occurring Ralstonia solanacearum- YPELAAALPELTRAHIVDIARQRSGDLAL derived protein N-terminus QALLPVATALTAAPLRLSASQIATVAQY GERPAIQALYRLRRKLTRAPLH SEQ ID NO: 303 Truncated N-terminus; positions 1 KQESLSEIAKYHTTLTGQGFTHADICRIS (H) to 120 (K) of the naturally RRRQSLRVVARNYPELAAALPELTRAHI occurring Ralstonia solanacearum- VDIARQRSGDLALQALLPVATALTAAPL derived protein N-terminus RLSASQIATVAQYGERPAIQALYRLRRK LTRAPLH SEQ ID NO: 265 Half-repeat LSTAQVVAIACISGQQALE SEQ ID NO: 266 Truncated C-terminus; positions 1 (A) AIEAHMPTLRQASHSLSPERVAAIACIGG to 63 (S) of the naturally occurring RSAVEAVRQGLPVKAIRRIRREKAPVAG Ralstonia solanacearum-derived PPPAS protein C-terminus

In some embodiments, an RNBD can be engineered to target and bind to a site in the PDCD1 gene. For example, an RNBD with the sequence FGKLVALGYSREQIRKLKQESLSEIAKYHTTLTGQGFTHADICRISRRRQSLRVVARNYPELA AALPELTRAHIVDIARQRSGDLALQALLPVATALTAAPLRLSASQIATVAQYGERPAIQALY RLRRKLTRAPLHLTPQQVVAIASNTGGKRALEAVCVQLPVLRAAPYRLSTEQVVAIASHDG GKQALEAVGAQLVALRAAPYALSTEQVVAIASHDGGKQALEAVGAQLVALRAAPYALST AQVVAIASNGGGKQALEGIGEQLLKLRTAPYGLSTEQVVAIASNKGGKQALEAVKAHLLDL LGAPYVLSTEQVVAIASNKGGKQALEAVKAHLLDLLGAPYVLSTEQVVAIASNKGGKQAL EAVKAHLLDLLGAPYVLSTEQVVVIANSIGGKQALEAVKVQLPVLRAAPYELSTEQVVAIA SHDGGKQALEAVGAQLVALRAAPYALSTEQVVVIANSIGGKQALEAVKVQLPVLRAAPYE LSTEQVVAIASNKGGKQALEAVKAHLLDLLGAPYVLSTAQVVAIASNGGGKQALEGIGEQL LKLRTAPYGLSTAQVVAIASNGGGKQALEGIGEQLLKLRTAPYGLSTAQVVAIASNGGGKQ ALEGIGEQLLKLRTAPYGLSTEQVVAIASHDGGKQALEAVGAQLVALRAAPYALSTEQVVA IASHDGGKQALEAVGAQLVALRAAPYALSTEQVVAIASHDGGKQALEAVGAQLVALRAAP YALSTAQVVAIASNGGGKQALEGIGEQLLKLRTAPYGLSTAQVVAIASNGGGKQALEGIGE QLLKLRTAPYGLSTAQVVAIACISGQQALEAIEAHMPTLRQASHSLSPERVAAIACIGGRSAV EAVRQGLPVKAIRRIRREKAPVAGPPPAS (SEQ ID NO: 311) can bind to the GACCTGGGACAGTTTCCCTT (SEQ ID NO: 312) nucleic acid sequence in the PDCD1 gene. As another example, an RNBD with the sequence FGKLVALGYSREQIRKLKQESLSEIAKYHTTLTGQGFTHADICRISRRRQSLRVVARNYPELA AALPELTRAHIVDIARQRSGDLALQALLPVATALTAAPLRLSASQIATVAQYGERPAIQALY RLRRKLTRAPLHLTPQQVVAIASNTGGKRALEAVCVQLPVLRAAPYRLSTAQVVAIASNGG GKQALEGIGEQLLKLRTAPYGLSTEQVVAIASHDGGKQALEAVGAQLVALRAAPYALSTA QVVAIASNGGGKQALEGIGEQLLKLRTAPYGLSTEQVVAIASHNGGKQALEAVKADLLELR GAPYALSTEQVVAIASHDGGKQALEAVGAQLVALRAAPYALSTEQVVVIANSIGGKQALEA VKVQLPVLRAAPYELSTAQVVAIASNGGGKQALEGIGEQLLKLRTAPYGLSTEQVVAIASH NGGKQALEAVKADLLELRGAPYALSTEQVVAIASHDGGKQALEAVGAQLVALRAAPYALS TEQVVAIASHDGGKQALEAVGAQLVALRAAPYALSTAQVVAIASNGGGKQALEGIGEQLL KLRTAPYGLSTEQVVAIASHNGGKQALEAVKADLLELRGAPYALSTEQVVAIASHNGGKQ ALEAVKADLLELRGAPYALSTEQVVVIANSIGGKQALEAVKVQLPVLRAAPYELSTEQVVA IASHNGGKQALEAVKADLLELRGAPYALSTEQVVAIASHDGGKQALEAVGAQLVALRAAP YALSTAQVVAIACISGQQALEMEAHMPTLRQASHSLSPERVAAIACIGGRSAVEAVRQGLP VKAIRRIRREKAPVAGPPPAS (SEQ ID NO: 313) can bind to the GATCTGCATGCCTGGAGC (SEQ ID NO: 314) nucleic acid sequence in the PDCD1 gene. As yet another example, an RNBD with the sequence FGKLVALGYSREQIRKLKQESLSEIAKYHTTLTGQGFTHADICRISRRRQSLRVVARNYPELA AALPELTRAHIVDIARQRSGDLALQALLPVATALTAAPLRLSASQIATVAQYGERPAIQALY RLRRKLTRAPLHLTPQQVVAIASNTGGKRALEAVCVQLPVLRAAPYRLSTAQVVAIASNGG GKQALEGIGEQLLKLRTAPYGLSTEQVVAIASHDGGKQALEAVGAQLVALRAAPYALSTA QVVAIASNGGGKQALEGIGEQLLKLRTAPYGLSTEQVVAIASHNGGKQALEAVKADLLELR GAPYALSTEQVVAIASHDGGKQALEAVGAQLVALRAAPYALSTAQVVAIATRSGGKQALE AVRAQLLDLRAAPYGLSTAQVVAIASNGGGKQALEGIGEQLLKLRTAPYGLSTEQVVAIAS HNGGKQALEAVKADLLELRGAPYALSTEQVVAIASHDGGKQALEAVGAQLVALRAAPYA LSTEQVVAIASHDGGKQALEAVGAQLVALRAAPYALSTAQVVAIASNGGGKQALEGIGEQ LLKLRTAPYGLSTEQVVAIASHNGGKQALEAVKADLLELRGAPYALSTEQVVAIASHNGGK QALEAVKADLLELRGAPYALSTAQVVAIATRSGGKQALEAVRAQLLDLRAAPYGLSTEQV VAIASHNGGKQALEAVKADLLELRGAPYALSTEQVVAIASHDGGKQALEAVGAQLVALRA APYALSTAQVVAIACISGQQALEMEAHMPTLRQASHSLSPERVAAIACIGGRSAVEAVRQG LPVKAIRRIRREKAPVAGPPPAS (SEQ ID NO: 315) can bind to the GATCTGCATGCCTGGAGC (SEQ ID NO: 314) nucleic acid sequence in the PDCD1 gene. Any one of SEQ ID NO: 311, SEQ ID NO; 313, or SEQ ID NO: 315 can be fused to any repression domain described herein (e.g., KRAB) to yield a gene repressor capable of repressing expression of the target gene.

Xanthomonas Derived Transcription Activator Like Effector (TALE)

The present disclosure provides a modular nucleic acid binding domain derived from Xanthomonas spp., also referred to herein as a transcription activator-like effector (TALE) protein, can comprise a plurality of repeat units. A repeat unit of the plurality of repeat units recognizes a single target nucleotide, base pair, or both. A repeat unit from Xanthomonas spp. can comprise 33-35 amino acid residues. In some embodiments, a repeat unit can be from Xanthomonas spp. protein having the sequence:

(SEQ ID NO: 299) MDPIRSRTPSPARELLPGPQPDGVQPTADRGVSPPAGGPLDGLPARRTMS RTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAA TGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPA AQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHP AALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRG PPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPV LCQAHGLTPQQVVAIASNSGGKQALETVQRLLPVLCQAHGLTPEQVVAIA SNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALL PVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVA IASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQR LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQV VAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETV QALLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAHGLTPE QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALE TVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLT PQQVVAIASNGGGRPALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALA ALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAD HAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEAR SGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLD APSPMHEGDQTRASSRKRSRSDRAVTGPSAQQSFEVRVPEQRDALHLPLS WRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGAADDTPAF NEEELAWLMELLPQ.

In some embodiments, a TALE of the present disclosure can comprise between 1 to 50 Xanthomonas spp.-derived repeat units. In some embodiments, a TALE of the present disclosure can comprise between 9 and 36 Xanthomonas spp.-derived repeat units. Preferably, in some embodiments, a TALE of the present disclosure can comprise between 12 and 30 Xanthomonas spp.-derived repeat units. A TALE described herein can comprise between 5 to 10 Xanthomonas spp.-derived repeat units, between 10 to 15 Xanthomonas spp.-derived repeat units, between 15 to 20 Xanthomonas spp.-derived repeat units, between 20 to 25 Xanthomonas spp.-derived repeat units, between 25 to 30 Xanthomonas spp.-derived repeat units, or between 30 to 35 Xanthomonas spp.-derived repeat units, between 35 to 40 Xanthomonas spp.-derived repeat units. A TALE described herein can comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or more Xanthomonas spp.-derived repeat units, such as, repeat units derived from Xanthomonas spp. protein having the amino acid sequence set forth in SEQ ID NO:299.

A Xanthomonas spp.-derived repeat units can be derived from a wild-type repeat unit, such as any one of SEQ ID NO: 323-SEQ ID NO: 326. For example, a Xanthomonas spp.-derived repeat units can have a sequence of LTPDQVVAIASNHGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 323) comprising an RVD of NH, which recognizes guanine. A Xanthomonas spp.-derived repeat units can have a sequence of LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 324) comprising an RVD of NG, which recognizes thymidine. A Xanthomonas spp.-derived repeat units can have a sequence of LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 325) comprising an RVD of NI, which recognizes adenosine. A Xanthomonas spp.-derived repeat units can have a sequence of LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 326) comprising an RVD of HD, which recognizes cytosine.

A Xanthomonas spp.-derived repeat unit can also comprise a modified Xanthomonas spp.-derived repeat units enhanced for specific recognition of a nucleotide or base pair. A TALE described herein can comprise one or more wild-type Xanthomonas spp.-derived repeat units, one or more modified Xanthomonas spp.-derived repeat units, or a combination thereof. In some embodiments, a modified Xanthomonas spp.-derived repeat units can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 mutations that can enhance recognition of a specific nucleotide or base pair. In some embodiments, a modified Xanthomonas spp.-derived repeat unit can comprise more than 1 modification, for example 1 to 5 modifications, 5 to 10 modifications, 10 to 15 modifications, 15 to 20 modifications, 20 to 25 modification, or 25-29 modifications. In some embodiments, A TALE can comprise more than one modified Xanthomonas spp.-derived repeat units, wherein each of the modified Xanthomonas spp.-derived repeat units can have a different number of modifications.

In some embodiments, a TALE of the present disclosure can have the full length naturally occurring N-terminus of a naturally occurring Xanthomonas spp.-derived protein, such as the N-terminus of SEQ ID NO: 299. The N-terminus sequence in SEQ ID NO:299 is indicated by underlining.

In some embodiments, a TALE of the present disclosure can comprise the amino acid residues at position 1 (N) through position 137 (M) of the naturally occurring Xanthomonas spp.-derived protein as follows:

(SEQ ID NO: 300) MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPA ALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGP PLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN.

The amino acid sequence set forth in SEQ ID NO:300 includes a M added to the N-terminus which is not present in the wild type N-terminus region of a TALE protein. The N-terminus fragment sequence set out in SEQ ID NO:300 is generated by deleting amino acids N+288 through N+137 of the N-terminus region of a TALE protein, adding a M, such that amino acids N+136 through N+1 of the N-terminus region of the TALE protein are present.

In some embodiments, the N-terminus can be truncated such that the fragment of the N-terminus includes amino acids from position 1 (N) through position 120 (K) of the naturally occurring Xanthomonas spp.-derived protein as follows:

(SEQ ID NO: 301) KPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALP EATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGG VTAVEAVHAWRNALTGAPLN.

In some embodiments, the N-terminus can be truncated such that the fragment of the N-terminus includes amino acids from position 1 (N) through position 115 (S) of the naturally occurring Xanthomonas spp.-derived protein as follows:

(SEQ ID NO: 321) STVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHE AIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVE AVHAWRNALTGAPLN.

In some embodiments, the N-terminus can be truncated such that the fragment of the N-terminus includes amino acids from position 1 (N) through position 110 (H) of the naturally occurring Xanthomonas spp.-derived protein as follows:

(SEQ ID NO: 447) HHEALVGHGETHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGV GKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAW RNALTGAPLN.

In some embodiments, a truncation of the naturally occurring Xanthomonas spp.-derived protein can be used at the N-terminus of a TALE disclosed herein. In some embodiments, a truncation of the naturally occurring Xanthomonas spp.-derived protein can be used at the N-terminus of a TALE disclosed herein and may include an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in one of SEQ ID NOs: 300, 301, 321, and 447. The naturally occurring N-terminus of Xanthomonas spp. can be truncated to amino acid residues at positions 1 to 50, 1 to 70, 1 to 100, 1 to 120, 1 to 130, 10 to 40, 60 to 100, or 100 to 120 and used at the N-terminus of the TALE.

FIGS. 1A-1C show schematics of the domain structure of a TALE protein (not drawn to scale). ‘N’ and ‘C’ indicate the amino and carboxy termini, respectively. The TALE repeat domain comprising TALE repeat units, N-Cap and C-Cap regions are labeled and the residue numbering scheme for the N-Cap and C-Cap regions and the N-terminus and C-terminus fragments are indicated. FIG. 1A includes the full-length N-cap region that extends from amino acid position N+1 to N+288 and full-length C-cap region that extends from amino acid position C+1 through C+278. FIG. 1B provides a schematic of a DNA binding protein comprising TALE repeat units and a truncated N-terminus that extends from amino acid position N+1 to N+136 (the notation N+137 indicates that a methionine added to the N-terminus increases the length to 137) and a truncated C-terminus that extends from amino acid position C+1 through C+63. FIG. 1C provides a schematic of a DNA binding protein comprising TALE repeat units and a truncated N-terminus that extends from amino acid position N+1 to N+115 and a truncated C-terminus that extends from amino acid position C+1 through C+63. In certain cases, the last repeat domain may be a half-repeat or a partial repeat as disclosed herein.

In some embodiments, a TALE of the present disclosure can have a DNA binding domain, in which the final full length repeat unit of 33-35 amino acid residues is followed by a half repeat also derived from Xanthomonas spp. The half repeat can have 15 to 23 amino acid residues, for example, the half repeat can have 19 amino acid residues. In particular embodiments, the half repeat can have a sequence as set forth in LTPQQVVAIASNGGGRPALE (SEQ ID NO: 297). In some embodiments, the half repeat can have a sequence as set forth in SEQ ID NO: 327, 328, 329, 330, 331, 332, 333, or 334).

TABLE 4 Xanthomonas Repeat Sequences SEQ ID NO Amino Acid Sequence Description 323 LTPDQVVAIASNHGGKQALETVQRLLPV RVD NH LCQDHG recognizing G 324 LTPDQVVAIASNGGGKQALETVQRLLPV RVD NG LCQDHG recognizing T 325 LTPDQVVAIASNIGGKQALETVQRLLPV RVD NI LCQDHG recognizing A 326 LTPDQVVAIASHDGGKQALETVQRLLPV RVD HD LCQDHG recognizing C 297 LTPQQVVAIASNGGGRPALE Half repeat 327 LTPEQVVAIASNGGGRPALE Half repeat 328 LTPDQVVAIASNGGGRPALE Half repeat 329 LTPEQVVAIASNIGGRPALE Half repeat 330 LTPDQVVAIASNIGGRPALE Half repeat 331 LTPEQVVAIASHDGGRPALE Half repeat 332 LTPDQVVAIASHDGGRPALE Half repeat 333 LTPEQVVAIASNHGGRPALE Half repeat 334 LTPDQVVAIASNHGGRPALE Half repeat

In some embodiments, a TALE of the present disclosure can have the full length naturally occurring C-terminus of a naturally occurring Xanthomonas spp.-derived protein, such as the C-terminus of SEQ ID NO: 299. The C-terminus of the TALE protein sequence set forth in SEQ ID NO:299 is italicized. In some embodiments, the C-terminus can be a fragment of the full length naturally occurring C-terminus of a naturally occurring Xanthomonas spp.-derived protein. In some embodiments, the C-terminus can be less than 250 amino acids long. In some embodiments, the C-terminus can be positions 1 (S) through position 278 (Q) of the naturally occurring Xanthomonas spp.-derived protein as follows: SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRV ADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPAS QRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRS RSDRAVTGPSAQQSFEVRAPEQRDALHLPLSWRVKRPRTSIGGGLPDPGTPTAADLAASSTV MREQDEDPFAGAADDFPAFNEEELAWLMELLPQ (SEQ ID NO: 302). In some embodiments, any truncation of the full length naturally occurring C-terminus of a naturally occurring Xanthomonas spp.-derived protein can be used at the C-terminus of a TALE of the present disclosure. For example, in some embodiments, the naturally occurring N-terminus of Xanthomonas spp. can be truncated to amino acid residues at position 1 (S) to position 63 (X) as follows: SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRV A (SEQ ID NO: 298). The naturally occurring C-terminus of Xanthomonas spp. can be truncated amino acid residues at positions 1 to 50 and used at the C-terminus of the engineered DNA binding domain. The naturally occurring C-terminus of Xanthomonas spp. can be truncated to amino acid residues at positions 1 to 63, 1 to 50, 1 to 70, 1 to 100, 1 to 120, 1 to 130, 10 to 40, 60 to 100, or 100 to 120 and used at the C-terminus of the engineered DNA binding domain.

The terms “N-cap” polypeptide and “N-terminal sequence” are used to refer to an amino acid sequence (polypeptide) that flanks the N-terminal portion of the first TALE repeat unit. The N-cap sequence can be of any length (including no amino acids), so long as the TALE-repeat unit(s) function to bind DNA. An N-terminal fragment and grammatical equivalents thereof refers to a shortened sequence of an N-terminal sequence which fragment is sufficient for the TALE repeat units to bind to DNA.

The term “C-cap” or “C-terminal region” refers to optionally present amino acid sequences that may be flanking the C-terminal portion of the last TALE repeat unit. The C-cap can also comprise any part of a terminal C-terminal TALE repeat, including 0 residues, truncations of a TALE repeat or a full TALE repeat. A C-terminal fragment and grammatical equivalents thereof refers to a shortened sequence of a C-terminal sequence which fragment is sufficient for the TALE repeat units to bind to DNA.

Animal Pathogen Derived Modular Nucleic Acid Binding Domains

The present disclosure provides a modular nucleic acid binding domain derived from an animal pathogen protein (MAP-NBD) can comprise a plurality of repeat units, wherein a repeat unit of the plurality of repeat units recognizes a single target nucleotide, base pair, or both.

In some embodiments, the repeat unit can be derived from an animal pathogen, and can be referred to as a non-naturally occurring modular nucleic acid binding domain derived from an animal pathogen protein (MAP-NBD), or “modular animal pathogen-nucleic acid binding domain” (MAP-NBD). For example, in some cases, the animal pathogen can be from the Gram-negative bacterium genus, Legionella. In other cases, the animal pathogen can be from Burkholderia. In some cases, the animal pathogen can be from Paraburkholderia. In other cases, the animal pathogen can be from Francisella.

In particular embodiments, the repeat unit can be derived from a species of the genus of Legionella, such as Legionella quateirensis, the genus of Burkholderia, the genus of Paraburkholderia, or the genus of Francisella. In some embodiments, the repeat unit can comprise from 19 amino acid residues to 35 amino acid residues. In particular embodiments, the repeat unit can comprise 33 amino acid residues. In other embodiments, the repeat unit can comprise 35 amino acid residues. In some embodiments, the MAP-NBD is non-naturally occurring, and comprises a plurality of repeat units and wherein a repeat unit of the plurality of repeat units recognizes a single target nucleic acid.

In some embodiments, a repeat unit can be derived from a Legionella quateirensis protein with the following sequence:

(SEQ ID NO: 281) MPDLELNFAIPLHLFDDETVFTHDATNDNSQASSSYSSKSSPASANARKR TSRKEMSGPPSKEPANTKSRRANSQNNKLSLADRLTKYNIDEEFYQTRSD SLLSLNYTKKQIERLILYKGRTSAVQQLLCKHEELLNLISPDGLGHKELI KIAARNGGGNNLIAVLSCYAKLKEMGFSSQQIIRMVSHAGGANNLKAVTA NHDDLQNMGFNVEQIVRMVSHNGGSKNLKAVTDNHDDLKNMGFNAEQIVR MVSHGGGSKNLKAVTDNHDDLKNMGFNAEQIVSMVSNNGGSKNLKAVTDN HDDLKNMGFNAEQIVSMVSNGGGSLNLKAVKKYHDALKDRGFNTEQIVRM VSHDGGSLNLKAVKKYHDALRERKFNVEQIVSIVSHGGGSLNLKAVKKYH DVLKDREFNAEQIVRMVSHDGGSLNLKAVTDNHDDLKNMGFNAEQIVRMV SHKGGSKNLALVKEYFPVFSSFHFTADQIVALICQSKQCFRNLKKNHQQW KNKGLSAEQIVDLILQETPPKPNFNNTSSSTPSPSAPSFFQGPSTPIPTP VLDNSPAPIFSNPVCFFSSRSENNTEQYLQDSTLDLDSQLGDPTKNFNVN NFWSLFPFDDVGYHPHSNDVGYHLHSDEESPFFDF.

In some embodiments, a repeat from a Legionella quateirensis protein can comprise a repeat with a canonical RVD or a non-canonical RVD. In some embodiments, a canonical RVD can comprise NN, NG, HD, or HD. In some embodiments, a non-canonical RVD can comprise RN, HA, HN, HG, HG, or HK.

In some embodiments, a repeat of SEQ ID NO: 282 comprises an RVD of HA and primarily recognizes a base of adenine (A). In some embodiments, a repeat of SEQ ID NO: 283 comprises an RVD of HN and recognizes a base comprising guanine (G). In some embodiments, a repeat of SEQ ID NO: 284 comprises an RVD of HG and recognizes a base comprising thymine (T). In some embodiments, a repeat of SEQ ID NO: 285 comprises an RVD of NN and recognizes a base comprising guanine (G). In some embodiments, a repeat of SEQ ID NO: 286 comprises an RVD of NG and recognizes a base comprising thymine (T). In some embodiments, a repeat of SEQ ID NO: 287 comprises an RVD of HD and recognizes a base comprising cytosine (C). In some embodiments, a repeat of SEQ ID NO: 288 comprises an RVD of HG and recognizes a base comprising thymine (T). In some embodiments, a repeat of SEQ ID NO: 289 comprises an RVD of HD and recognizes a base comprising cytosine (C). In some embodiments, a half-repeat of SEQ ID NO: 290 comprises an RVD of HK and recognizes a base comprising guanine (G). In some embodiments, a repeat of SEQ ID NO: 357 comprises an RVD of RN and recognizes a base comprising guanine (G).

TABLE 5 illustrates exemplary repeats from Legionella quateirensis, Burkholderia, Paraburkholderia, or Francisella that can make up a MAP-NBD of the present disclosure and the RVD at position 12 and 13 of the particular repeat. A MAP-NBD of the present disclosure can comprise at least one of the repeats disclosed in TABLE 5 including any one of SEQ ID NO: 357, SEQ ID NO: 282-SEQ ID NO: 290, or SEQ ID NO: 358-SEQ ID NO: 446. A MAP-NBD of the present disclosure can comprise any combination of repeats disclosed in TABLE 5 including any one of SEQ ID NO: 357, SEQ ID NO: 282-SEQ ID NO: 290, or SEQ ID NO: 358-SEQ ID NO: 446.

TABLE 5 Animal Pathogen Derived Repeat Units SEQ ID NO Organism Repeat Unit Sequence RVD 357 L. quateirensis LGHKELIKIAARNGGGNNLIAVLSCYAKLKEMG RN 282 L. quateirensis FSSQQIIRMVSHAGGANNLKAVTANHDDLQNMG HA 283 L. quateirensis FNVEQIVRMVSHNGGSKNLKAVTDNHDDLKNMG HN 284 L. quateirensis FNAEQIVRMVSHGGGSKNLKAVTDNHDDLKNMG HG 285 L. quateirensis FNAEQIVSMVSNNGGSKNLKAVTDNHDDLKNMG NN 286 L. quateirensis FNAEQIVSMVSNGGGSLNLKAVKKYHDALKDRG NG 287 L. quateirensis FNTEQIVRMVSHDGGSLNLKAVKKYHDALRERK HD 288 L. quateirensis FNVEQIVSIVSHGGGSLNLKAVKKYHDVLKDRE HG 289 L. quateirensis FNAEQIVRMVSHDGGSLNLKAVTDNHDDLKNMG HD 290 L. quateirensis FNAEQIVRMVSHKGGSKNL HK (half repeat) 358 L. quateirensis FSAEQIVRIAAHDGGSRNIEAVQQAQHVLKELG HD 359 L. quateirensis FSAEQIVSIVAHDGGSRNIEAVQQAQHILKELG HD 360 L. quateirensis FSRQQILRIASHDGGSKNIAAVQKFLPKLMNFGFN HD 361 L. quateirensis FSAEQIVRIAAHDGGSLNIDAVQQAQQALKELG HD 362 L. quateirensis FSTEQIVCIAGHGGGSLNIKAVLLAQQALKDLG HG 363 L. quateirensis FSSEQIVRVAAHGGGSLNIKAVLQAHQALKELD HG 364 L. quateirensis FSAEQIVHIAAHGGGSLNIKAILQAHQTLKELN HG 365 L. quateirensis FSAEQIVRIAAHIGGSRNIEAIQQAHHALKELG HI 366 L. quateirensis FSAEQIVRIAAHIGGSHNLKAVLQAQQALKELD HI 367 L. quateirensis FSAKHIVRIAAHIGGSLNIKAVQQAQQALKELG HI 368 L. quateirensis FNAEQIVRMVSHKGGSKNLALVKEYFPVFSSFH HK 369 L. quateirensis FNAEQIVRMVSHKGGSKNLALVKEYFPVFSSFHFT HK 370 L. quateirensis FSADQIVRIAAHKGGSHNIVAVQQAQQALKELD HK 371 L. quateirensis FNVEQIVRMVSHNGGSKNLKAVTDNHDDLKNMGFN HN 372 L. quateirensis FSADQVVKIAGHSGGSNNIAVMLAVFPRLRDFGFK HS 373 L. quateirensis FSAEQIVSIAAHVGGSHNIEAVQKAHQALKELD HV 374 L. quateirensis FNAEQIVSMVSNNGGSKNLKAVTDNHDDLKNMGFN NN 375 L. quateirensis FSHKELIKIAARNGGGNNLIAVLSCYAKLKEMG RN 376 L. quateirensis FSHKELIKIAARNGGGNNLIAVLSCYAKLKEMGFS RN 377 Burkholderia FSSGETVGATVGAGGTETVAQGGTASNTTVSSGGY GA 378 Burkholderia FSGGMATSTTVGSGGTQDVLAGGAAVGGTVGTGGV GS 379 Burkholderia FSAADIVKIAGKIGGAQALQAFITHRAALIQAGFS KI 380 Burkholderia FNPTDIVKIAGNDGGAQALQAVLELEPALRERGFS ND 381 Burkholderia FNPTDIVRMAGNDGGAQALQAVFELEPAFRERSFS ND 382 Burkholderia FNPTDIVRMAGNDGGAQALQAVLELEPAFRERGFS ND 383 Burkholderia FSQVDIVKIASNDGGAQALYSVLDVEPTFRERGFS ND 384 Burkholderia FSRADIVKIAGNDGGAQALYSVLDVEPPLRERGFS ND 385 Burkholderia FSRGDIVKIAGNDGGAQALYSVLDVEPPLRERGFS ND 386 Burkholderia FNRADIVRIAGNGGGAQALYSVRDAGPTLGKRGFS NG 387 Burkholderia FRQADIVKIASNGGSAQALNAVIKLGPTLRQRGFS NG 388 Burkholderia FRQADIVKMASNGGSAQALNAVIKLGPTLRQRGFS NG 389 Burkholderia FSRADIVKIAGNGGGAQALQAVLELEPTFRERGFS NG 390 Burkholderia FSRADIVRIAGNGGGAQALYSVLDVGPTLGKRGFS NG 391 Burkholderia FSRGDIVRIAGNGGGAQALQAVLELEPTLGERGFS NG 392 Burkholderia FSRADIVKIAGNGGGAQALQAVITHRAALTQAGFS NG 393 Burkholderia FSRGDTVKIAGNIGGAQALQAVLELEPTLRERGFS NI 394 Burkholderia FNPTDIVKIAGNIGGAQALQAVLELEPAFRERGFS NI 395 Burkholderia FSAADIVKIAGNIGGAQALQAIFTHRAALIQAGFS NI 396 Burkholderia FSAADIVKIAGNIGGAQALQAVITHRATLTQAGFS NI 397 Burkholderia FSATDIVKIASNIGGAQALQAVISRRAALIQAGFS NI 398 Burkholderia FSQPDIVKIAGNIGGAQALQAVLELEPAFRERGFS NI 399 Burkholderia FSRADIVKIAGNIGGAQALQAVLELESTFRERSFN NI 400 Burkholderia FSRADIVKIAGNIGGAQALQAVLELESTLRERSFN NI 401 Burkholderia FSRGDIVKMAGNIGGAQALQAGLELEPAFRERGFS NI 402 Burkholderia FSRGDIVKMAGNIGGAQALQAVLELEPAFHERSFC NI 403 Burkholderia FTLTDIVKMAGNIGGAQALKAVLEHGPTLRQRDLS NI 404 Burkholderia FTLTDIVKMAGNIGGAQALKVVLEHGPTLRQRDLS NI 405 Burkholderia FNPTDIVKIAGNNGGAQALQAVLELEPALRERGFS NN 406 Burkholderia FNPTDIVKIAGNNGGAQALQAVLELEPALRERSFS NN 407 Burkholderia FNPTDMVKIAGNNGGAQALQAVLELEPALRERGFS NN 408 Burkholderia FSAADIVKIASNNGGAQALQALIDHWSTLSGKTKA NN 409 Burkholderia FSAADIVKIASNNGGAQALQAVISRRAALIQAGFS NN 410 Burkholderia FSAADIVKIASNNGGAQALQAVITHRAALAQAGFS NN 411 Burkholderia FSAADIVKIASNNGGARALQALIDHWSTLSGKTKA NN 412 Burkholderia FTLTDIVEMAGNNGGAQALKAVLEHGSTLDERGFT NN 413 Burkholderia FTLTDIVKMAGNNGGAQALKAVLEHGPTLDERGFT NN 414 Burkholderia FTLTDIVKMAGNNGGAQALKVVLEHGPTLRQRGFS NN 415 Burkholderia FTLTDIVKMASNNGGAQALKAVLEHGPTLDERGFT NN 416 Burkholderia FSAADIVKIAGNSGGAQALQAVISHRAALTQAGFS NS 417 Burkholderia FSGGDAVSTVVRSGGAQSVASGGTASGTTVSAGAT RS 418 Burkholderia FRQTDIVKMAGSGGSAQALNAVIKHGPTLRQRGFS SG 419 Burkholderia FSLIDIVEIASNGGAQALKAVLKYGPVLTQAGRS SN 420 Burkholderia FSGGDAAGTVVSSGGAQNVTGGLASGTTVASGGAA SS 421 Paraburkholderia FNLTDIVEMAANSGGAQALKAVLEHGPTLRQRGLS NS 422 Paraburkholderia FNRASIVKIAGNSGGAQALQAVLKHGPTLDERGFN NS 423 Paraburkholderia FSQANIVKMAGNSGGAQALQAVLDLELVFRERGFS NS 424 Paraburkholderia FSQPDIVKMAGNSGGAQALQAVLDLELAFRERGFS NS 425 Paraburkholderia FSLIDIVEIASNGGAQALKAVLKYGPVLMQAGRS SN 426 Francisella YKSEDIIRLASHDGGSVNLEAVLRLHSQLTRLG HD 427 Francisella YKPEDIIRLASHGGGSVNLEAVLRLNPQLIGLG HG 428 Francisella YKSEDIIRLASHGGGSVNLEAVLRLHSQLTRLG HG 429 Francisella YKSEDIIRLASHGGGSVNLEAVLRLNPQLIGLG HG 430 Paraburkholderia FNLTDIVEMAGKGGGAQALKAVLEHGPTLRQRGFN KG 431 Paraburkholderia FRQADIIKIAGNDGGAQALQAVIEHGPTLRQHGFN ND 432 Paraburkholderia FSQADIVKIAGNDGGTQALHAVLDLERMLGERGFS ND 433 Paraburkholderia FSRADIVKIAGNGGGAQALKAVLEHEATLDERGFS NG 434 Paraburkholderia FSRADIVRIAGNGGGAQALYSVLDVEPTLGKRGFS NG 435 Paraburkholderia FSQPDIVKMASNIGGAQALQAVLELEPALRERGFS NI 436 Paraburkholderia FSQPDIVKMAGNIGGAQALQAVLSLGPALRERGFS NI 437 Paraburkholderia FSQPEIVKIAGNIGGAQALHTVLELEPTLHKRGFN NI 438 Paraburkholderia FSQSDIVKIAGNIGGAQALQAVLDLESMLGKRGFS NI 439 Paraburkholderia FSQSDIVKIAGNIGGAQALQAVLELEPTLRESDFR NI 440 Paraburkholderia FNPTDIVKIAGNKGGAQALQAVLELEPALRERGFN NK 441 Paraburkholderia FSPTDIIKIAGNNGGAQALQAVLDLELMLRERGFS NN 442 Paraburkholderia FSQADIVKIAGNNGGAQALYSVLDVEPTLGKRGFS NN 443 Paraburkholderia FSRGDIVTIAGNNGGAQALQAVLELEPTLRERGFN NN 444 Paraburkholderia FSRIDIVKIAANNGGAQALHAVLDLGPTLRECGFS NN 445 Paraburkholderia FSQADIVKIVGNNGGAQALQAVFELEPTLRERGFN NN 446 Paraburkholderia FSQPDIVRITGNRGGAQALQAVLALELTLRERGFS NR

In any one of the animal pathogen-derived repeat domains of SEQ ID NO: 357, SEQ ID NO: 282-SEQ ID NO: 290, and SEQ ID NO: 358-SEQ ID NO: 446, there can be considerable sequence divergence between repeats of a MAP-NBD outside of the RVD.

In some embodiments, a MAP-NBD of the present disclosure can comprise between 1 to 50 animal pathogen-derived repeat units. In some embodiments, a MAP-NBD of the present disclosure can comprise between 9 and 36 animal pathogen-derived repeat units. In some embodiments, a MAP-NBD of the present disclosure can comprise between 12 and 30 animal pathogen-derived repeat units. A MAP-NBD described herein can comprise between 5 to 10, 10 to 15, 15-20, 20 to 25, 25 to 30, 30 to 35, or 35 to 40, e.g., 15-25 animal pathogen-derived repeat units. A MAP-NBD described herein can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 animal pathogen-derived repeat units.

A MAP-NBD described herein can comprise 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 animal pathogen-derived repeat units.

An animal pathogen-derived repeat units can be derived from a wild-type repeat unit, such as any one of SEQ ID NO: 357, SEQ ID NO: 282-SEQ ID NO: 290, and SEQ ID NO: 358-SEQ ID NO: 446. An animal pathogen-derived repeat unit can also comprise a modified animal pathogen-derived repeat units enhanced for specific recognition of a nucleotide or base pair. A MAP-NBD described herein can comprise one or more wild-type animal pathogen-derived repeat units, one or more modified animal pathogen-derived repeat units, or a combination thereof. In some embodiments, a modified animal pathogen-derived repeat units can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 mutations that can enhance recognition of a specific nucleotide or base pair. In some embodiments, a modified animal pathogen-derived repeat unit can comprise more than 1 modification, for example 1 to 5 modifications, 5 to 10 modifications, 10 to 15 modifications, 15 to 20 modifications, 20 to 25 modification, or 25-29 modifications. In some embodiments, a MAP-NBD can comprise more than one modified animal pathogen-derived repeat units, wherein each of the modified animal pathogen-derived repeat units can have a different number of modifications.

In some embodiments, a MAP-NBD of the present disclosure can have the full length naturally occurring N-terminus of a naturally occurring Legionella quateirensis-derived protein, such as the N-terminus of SEQ ID NO: 281. A N-terminus can be the full length N-terminus sequence and can have a sequence of MPDLELNFAIPLHLFDDETVFTHDATNDNSQASSSYSSKSSPASANARKRTSRKEMSGPPSK EPANTKSRRANSQNNKLSLADRLTKYNIDEEFYQTRSDSLLSLNYTKKQIERLILYKGRTSA VQQLLCKHEELLNLISPDG (SEQ ID NO: 291). In some embodiments, any truncation of SEQ ID NO: 291 can be used as the N-terminus in a MAP-NBD of the present disclosure. For example, in some embodiments, a MAP-NBD comprises a truncated N-terminus including amino acid residues at position 1 (G) to position 137 (S) of the naturally occurring Legionella quateirensis N-terminus as follows: NFAIPLHLFDDETVFTHDATNDNSQASSSYSSKSSPASANARKRTSRKEMSGPPSKEPANTK SRRANSQNNKLSLADRLTKYNIDEEFYQTRSDSLLSLNYTKKQIERLILYKGRTSAVQQLLC KHEELLNLISPDG (SEQ ID NO: 335). For example, in some embodiments, a MAP-NBD comprises a truncated N-terminus including amino acid residues at position 1 (G) to position 120 (S) of the naturally occurring Legionella quateirensis N-terminus as follows: DATNDNSQASSSYSSKSSPASANARKRTSRKEMSGPPSKEPANTKSRRANSQNNKLSLADR LTKYNIDEEFYQTRSDSLLSLNYTKKQIERLILYKGRTSAVQQLLCKHEELLNLISPDG (SEQ ID NO: 304). In some embodiments, a MAP-NBD comprises a truncated N-terminus including amino acid residues at position 1 (G) to position 115 (K) of the naturally occurring Legionella quateirensis N-terminus as follows: NSQASSSYSSKSSPASANARKRTSRKEMSGPPSKEPANTKSRRANSQNNKLSLADRLTKYNI DEEFYQTRSDSLLSLNYTKKQIERLILYKGRTSAVQQLLCKHEELLNLISPDG (SEQ ID NO: 322). In some embodiments, any truncation of the naturally occurring Legionella quateirensis-derived protein can be used at the N-terminus of a DNA binding domain disclosed herein. The naturally occurring N-terminus of Legionella quateirensis can be truncated to amino acid residues at positions 1 to 50, 1 to 70, 1 to 100, 1 to 120, 1 to 130, 10 to 40, 60 to 100, or 100 to 120 and used at the N-terminus of the MAP-NBD.

In some embodiments, a MAP-NBD of the present disclosure can have the full length naturally occurring C-terminus of a naturally occurring Legionella quateirensis-derived protein. In some embodiments, A MAP-NBD of the present disclosure can have at its C-terminus amino acid residues at position 1 (A) to position 176 (F) of the naturally occurring Legionella quateirensis-derived protein as follows:

(SEQ ID NO: 305) ALVKEYFPVFSSFHFTADQIVALICQSKQCFRNLKKNHQQWKNKGLSAEQ IVDLILQETPPKPNFNNTSSSTPSPSAPSFFQGPSTPIPTPVLDNSPAPI FSNPVCFFSSRSENNTEQYLQDSTLDLDSQLGDPTKNFNVNNFWSLFPFD DVGYHPHSNDVGYHLHSDEESPFFDF.

In some embodiments, a MAP-NBD of the present disclosure can have at its C-terminus amino acid residues at position 1 (A) to position 63 (P) of the naturally occurring Legionella quateirensis-derived protein as follows: ALVKEYFPVFSSFHFTADQIVALICQSKQCFRNLKKNHQQWKNKGLSAEQIVDLILQETPPK P (SEQ ID NO: 306).

In some embodiments, the present disclosure provides methods for identifying an animal pathogen-derived repeat unit. For example, a consensus sequence can be defined comprising a first repeat motif, a spacer, and a second repeat motif. The consensus sequence can be 1xxx211x1xxx33x2x1xxxxxxxxx1xxxx1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 292), 1xxx211x1xxx33x2x1xxxxxxxxx1xxxxx1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 293), 1xxx211x1xxx33x2x1xxxxxxxxx1xxxxxx1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 294), 1xxx211x1xxx33x2x1xxxxxxxxx1xxxxxxx1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 295), 1xxx211x1xxx33x2x1xxxxxxxxx1xxxxxxxx1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 296). For any one of SEQ ID NO: 292-SEQ ID NO: 296, x can be any amino acid residue, 1, 2, and 3 are flexible residues that are defined as follows: 1 can be selected from any one of A, F, I, L, M, T, or V, 2 can be selected from any one of D, E, K, N, M, S, R, or Q, and 3 can be selected from any one of A, G, N, or S. Thus, in some embodiments, a MAP-NBD can be derived from an animal pathogen comprising the consensus sequence of SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, or SEQ ID NO: 296. Any one of consensus sequences of SEQ ID NO: 292-SEQ ID NO: 296 can be compared against all sequences downloaded from NCBI, MGRast, JGI, and EBI databases to identify matches corresponding to animal pathogen proteins containing repeat units of a DNA-binding repeat unit.

In some embodiments, a MAP-NBD repeat unit can itself have a consensus sequence of 1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 293), wherein x can be any amino acid residue, 1, 2, and 3 are flexible residues that are defined as follows: 1 can be selected from any one of A, F, I, L, M, T, or V, 2 can be selected from any one of D, E, K, N, M, S, R, or Q, and 3 can be selected from any one of A, G, N, or S.

Mixed DNA Binding Domains

In some embodiments, the present disclosure provides DNA binding domains in which the repeat units, the N-terminus, and the C-terminus can be derived from any one of Ralstonia solanacearum, Xanthomonas spp., Legionella quateirensis, Burkholderia, Paraburkholderia, or Francisella. For example, the present disclosure provides a DNA binding domain wherein the plurality of repeat units are selected from any one of SEQ ID NO: 168-SEQ ID NO: 263 or SEQ ID NO: 336-SEQ ID NO: 356 and can further comprise an N-terminus and/or C-terminus from Xanthomonas spp., (N-termini: SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 301, and SEQ ID NO: 321; C-termini: SEQ ID NO: 302 and SEQ ID NO: 298) or Legionella quateirensis (N-termini: SEQ ID NO: 304 or SEQ ID NO: 322; C-termini: SEQ ID NO: 305 and SEQ ID NO: 306). In some embodiments, the present disclosure provides modular DNA binding domains in which the repeat units can be from Ralstonia solanacearum (e.g., any one of SEQ ID NO: 168-SEQ ID NO: 263 or SEQ ID NO: 336-SEQ ID NO: 356), Xanthomonas spp. (e.g., any one of SEQ ID NO: 323-SEQ ID NO: 334), an animal pathogen such as Legionella quateirensis, Burkholderia, Paraburkholderia, or Francisella (e.g., any one of SEQ ID NO: 357, SEQ ID NO: 282-SEQ ID NO: 290, or SEQ ID NO: 358-SEQ ID NO: 446), or any combination thereof.

Nucleases for Genome Editing

Genome editing can include the process of modifying a DNA of a cell in order to introduce or knock out a target gene or a target gene region. In some instances, a subject may have a disease in which a protein is aberrantly expressed or completely lacking. One therapeutic strategy for treating this disease can be introduction of a target gene or a target gene region to correct the aberrant or missing protein. For example, genome editing can be used to modify the DNA of a cell in the subject in order to introduce a functional gene, which gives rise to a functional protein. Introduction of this functional gene and expression of the functional protein can relieve the disease state of the subject.

In other instances, a subject may have a disease in which protein is overexpressed or is targeted by a virus for infection of a cell. Alternatively, a therapy such as a cell therapy for cancer can be ineffective due to repression of certain processes by tumor cells (e.g., checkpoint inhibition). Still alternatively, it may be desirable to eliminate a particular protein expressed at the surface of a cell in order to generate a universal, off-the-shelf cell therapy for a subject in need thereof (e.g., TCR). In such cases, it can be desirable to partially or completely knock out the gene encoding for such a protein. Genome editing can be used to modify the DNA of a cell in the subject in order to partially or completely knock out the target gene, thus reducing or eliminating expression of the protein of interest.

Genome editing can include the use of any nuclease as described herein in combination with any DNA binding domain disclosed herein in order to bind to a target gene or target gene region and induce a double strand break, mediated by the nuclease. Genes can be introduced during this process, or DNA binding domains can be designed to cut at regions of the DNA such that after non-homologous end joining, the target gene or target gene region is removed. Genome editing systems that are further disclosed and described in detail herein can include DNA binding domains from Xanthomonas, Ralstonia, Legionella, Burkholderia, Paraburkholderia, or Francisella fused to nucleases.

The specificity and efficiency of genome editing can be dependent on the nuclease responsible for cleavage. More than 3,000 type II restriction endonucleases have been identified. They recognize short, usually palindromic, sequences of 4-8 bp and, in the presence of Mg2+, cleave the DNA within or in close proximity to the recognition sequence. Naturally, type IIs restriction enzymes themselves have a DNA recognition domain that can be separated from the catalytic, or cleavage, domain. As such, since cleavage occurs at a site adjacent to the DNA sequence bound by the recognition domain, these enzymes can be referred to as exhibiting “shifted” cleavage. These type IIs restriction enzymes having both the recognition domain and the cleavage domain can be 400-600 amino acids. The main criterion for classifying a restriction endonuclease as a type II enzyme is that it cleaves specifically within or close to its recognition site and that it does not require ATP hydrolysis for its nucleolytic activity. An example of a type II restriction endonucleases is FokI, which consists of a DNA recognition domain and a non-specific DNA cleavage domain. FokI cleaves DNA nine and thirteen bases downstream of an asymmetric sequence (recognizing a DNA sequence of GGATG).

In some embodiments, the DNA cleavage domain at the C-terminus of FokI itself can be combined with a variety of DNA-binding domains (e.g., RNBDs, TALEs, MAP-NBDs) of other molecules for genome editing purposes. This cleavage domain can be 180 amino acids in length and can be directly linked to a DNA binding domain (e.g., RNBDs, TALEs, MAP-NBDs). In some embodiments, the FokI cleavage domain only comprises a single catalytic site. Thus, in order to cleave phosphodiester bonds, these enzymes form transient homodimers, providing two catalytic sites capable of cleaving double stranded DNA. In some embodiments, a single DNA-binding domains (e.g., RNBDs, TALEs, MAP-NBDs) linked to a Type IIS cleaving domain may not nick the double stranded DNA at the targeted site. In some embodiments, cleaving of target DNA only occurs when a pair of DNA-binding domains (e.g., RNBDs, TALEs, MAP-NBDs), each linked to a Type IIS cleaving domain (e.g., any one of SEQ ID NO: 1-SEQ ID NO: 81 (nucleotide sequences of SEQ ID NO: 82-SEQ ID NO: 162)) bind to opposing strands of DNA and allow for formation of a transient homodimer in the spacer region (the base pairs between the C-terminus of the DNA binding domain on a top strand of DNA and the C-terminus of the DNA binding domain on a bottom strand of DNA). Said spacer region can be greater than 2 base pairs, greater than 5 base pairs, greater than 10 base pairs, greater than 15 base pairs, greater than 24 base pairs, greater than 25 base pairs, greater than 30 base pairs, greater than 35 base pairs, greater than 40 base pairs, greater than 45 base pairs, or greater than 50 base pairs. In some embodiments, the spacer region can be anywhere from 2 to 50 base pairs, 5 to 40 base pairs, 10 to 30 base pairs, 14 to 40 base pairs, 24 to 30 base pairs, 24 to 40 base pairs, or 24 to 50 base pairs. In some embodiments, the nuclease disclosed herein (e.g., any one of SEQ ID NO: 1-SEQ ID NO: 81 (nucleotide sequences of SEQ ID NO: 82-SEQ ID NO: 162) can be capable of cleaving over a spacer region of greater than 24 base pairs upon formation of a transient homodimer.

Comparative analyses showed that FokI phylogenetic groupings can largely be at least partially explained by a combination of local gene duplication, and the whole-genome duplication event that predates their speciation, however enzymes vary significantly in their activities. In some aspects, the disclosure provides enzymes identified in a phylogenetic, molecular, and comparative analyses of sequences from various proteins related to FokI in various sequenced species. In some instances, such enzymes can comprise one or more mutations relative to SEQ ID NO: 1-SEQ ID NO: 81 (nucleotide sequences of SEQ ID NO: 82-SEQ ID NO: 162). In some cases, the non-naturally occurring enzymes described herein can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. A mutation can be engineered to enhance cleavage efficiency. A mutation can abolish cleavage activity. In some cases, a mutation can enhance homodimerization. For example, FokI can have a mutation at one or more amino acid residue positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 to modulate homodimerization, and similar mutations can be designed based on the phylogenetic analysis of SEQ ID NO: 1-SEQ ID NO: 81 (nucleotide sequences of SEQ ID NO: 82-SEQ ID NO: 162).

TABLE 6 shows exemplary amino acid sequences (SEQ ID NO: 1-SEQ ID NO: 81) of endonucleases for genome editing and the corresponding back-translated nucleic acid sequences (SEQ ID NO: 82-SEQ ID NO: 162) of the endonucleases, which were obtained using Genius software and selecting for human codon optimization.

TABLE 6 Amino Acid and Nucleic Acid Sequences of Endonucleases SEQ SEQ ID ID NO Amino Acid Sequence NO Back Translated Nucleic Acid Sequences 1 FLVKGAMEIKKSEL 82 TTCCTGGTGAAGGGCGCCATGGAGATCAAGAAGAGCGAGCTGA RHKLRHVPHEYIELI GGCACAAGCTGAGGCACGTGCCCCACGAGTACATCGAGCTGATC EIAQDSKQNRLLEFK GAGATCGCCCAGGACAGCAAGCAGAACAGGCTGCTGGAGTTCA VVEFFKKIYGYRGK AGGTGGTGGAGTTCTTCAAGAAGATCTACGGCTACAGGGGCAA HLGGSRKPDGALFT GCACCTGGGCGGCAGCAGGAAGCCCGACGGCGCCCTGTTCACC DGLVLNHGIILDTKA GACGGCCTGGTGCTGAACCACGGCATCATCCTGGACACCAAGGC YKDGYRLPISQADE CTACAAGGACGGCTACAGGCTGCCCATCAGCCAGGCCGACGAG MQRYVDENNKRSQ ATGCAGAGGTACGTGGACGAGAACAACAAGAGGAGCCAGGTGA VINPNEWWEIYPTSI TCAACCCCAACGAGTGGTGGGAGATCTACCCCACCAGCATCACC TDFKFLFVSGFFQGD GACTTCAAGTTCCTGTTCGTGAGCGGCTTCTTCCAGGGCGACTAC YRKQLERVSHLTKC AGGAAGCAGCTGGAGAGGGTGAGCCACCTGACCAAGTGCCAGG QGAVMSVEQLLLGG GCGCCGTGATGAGCGTGGAGCAGCTGCTGCTGGGCGGCGAGAA EKIKEGSLTLEEVGK GATCAAGGAGGGCAGCCTGACCCTGGAGGAGGTGGGCAAGAAG KFKNDEIVF TTCAAGAACGACGAGATCGTGTTC 2 QIVKSSIEMSKANM 83 CAGATCGTGAAGAGCAGCATCGAGATGAGCAAGGCCAACATGA RDNLQMLPHDYIELI GGGACAACCTGCAGATGCTGCCCCACGACTACATCGAGCTGATC EISQDPYQNRIFEMK GAGATCAGCCAGGACCCCTACCAGAACAGGATCTTCGAGATGA VMDLFINEYGFSGS AGGTGATGGACCTGTTCATCAACGAGTACGGCTTCAGCGGCAGC HLGGSRKPDGAMY CACCTGGGCGGCAGCAGGAAGCCCGACGGCGCCATGTACGCCC AHGFGVIVDTKAYK ACGGCTTCGGCGTGATCGTGGACACCAAGGCCTACAAGGACGG DGYNLPISQADEME CTACAACCTGCCCATCAGCCAGGCCGACGAGATGGAGAGGTAC RYVRENIDRNEHVN GTGAGGGAGAACATCGACAGGAACGAGCACGTGAACAGCAACA SNRWWNIFPEDTNE GGTGGTGGAACATCTTCCCCGAGGACACCAACGAGTACAAGTTC YKFLFVSGFFKGNFE CTGTTCGTGAGCGGCTTCTTCAAGGGCAACTTCGAGAAGCAGCT KQLERISIDTGVQGG GGAGAGGATCAGCATCGACACCGGCGTGCAGGGCGGCGCCCTG ALSVEHLLLGAEYIK AGCGTGGAGCACCTGCTGCTGGGCGCCGAGTACATCAAGAGGG RGILTLYDFKNSFLN GCATCCTGACCCTGTACGACTTCAAGAACAGCTTCCTGAACAAG KEIQF GAGATCCAGTTC 3 QTIKSSIEELKSELRT 84 CAGACCATCAAGAGCAGCATCGAGGAGCTGAAGAGCGAGCTGA QLNVISHDYLQLVDI GGACCCAGCTGAACGTGATCAGCCACGACTACCTGCAGCTGGTG SQDSQQNRLFEMKV GACATCAGCCAGGACAGCCAGCAGAACAGGCTGTTCGAGATGA MDLFINEFGYNGSH AGGTGATGGACCTGTTCATCAACGAGTTCGGCTACAACGGCAGC LGGSRKPDGILYTEG CACCTGGGCGGCAGCAGGAAGCCCGACGGCATCCTGTACACCG LSKDYGIIVDTKAYK AGGGCCTGAGCAAGGACTACGGCATCATCGTGGACACCAAGGC DGYNLPIAQADEME CTACAAGGACGGCTACAACCTGCCCATCGCCCAGGCCGACGAG RYIRENIDRNEVVNP ATGGAGAGGTACATCAGGGAGAACATCGACAGGAACGAGGTGG NRWWEVFPSKINDY TGAACCCCAACAGGTGGTGGGAGGTGTTCCCCAGCAAGATCAAC KFLFVSAYFKGNFK GACTACAAGTTCCTGTTCGTGAGCGCCTACTTCAAGGGCAACTT EQLERISINTGILGGA CAAGGAGCAGCTGGAGAGGATCAGCATCAACACCGGCATCCTG ISVEHLLLGAEYFKR GGCGGCGCCATCAGCGTGGAGCACCTGCTGCTGGGCGCCGAGTA GILSLEDVRDKFCNT CTTCAAGAGGGGCATCCTGAGCCTGGAGGACGTGAGGGACAAG EIEF TTCTGCAACACCGAGATCGAGTTC 4 GKSEVETIKEQMRG 85 GGCAAGAGCGAGGTGGAGACCATCAAGGAGCAGATGAGGGGCG ELTHLSHEYLGLLDL AGCTGACCCACCTGAGCCACGAGTACCTGGGCCTGCTGGACCTG AYDSKQNRLFELKT GCCTACGACAGCAAGCAGAACAGGCTGTTCGAGCTGAAGACCA MQLLTEECGFEGLH TGCAGCTGCTGACCGAGGAGTGCGGCTTCGAGGGCCTGCACCTG LGGSRKPDGIVYTK GGCGGCAGCAGGAAGCCCGACGGCATCGTGTACACCAAGGACG DENEQVGKENYGIII AGAACGAGCAGGTGGGCAAGGAGAACTACGGCATCATCATCGA DTKAYSGGYSLPISQ CACCAAGGCCTACAGCGGCGGCTACAGCCTGCCCATCAGCCAGG ADEMERYIGENQTR CCGACGAGATGGAGAGGTACATCGGCGAGAACCAGACCAGGGA DIRINPNEWWKNFG CATCAGGATCAACCCCAACGAGTGGTGGAAGAACTTCGGCGAC DGVTEYYYLFVAGH GGCGTGACCGAGTACTACTACCTGTTCGTGGCCGGCCACTTCAA FKGKYQEQIDRINCN GGGCAAGTACCAGGAGCAGATCGACAGGATCAACTGCAACAAG KNIKGAAVSIQQLLR AACATCAAGGGCGCCGCCGTGAGCATCCAGCAGCTGCTGAGGA IVNDYKAGKLTHED TCGTGAACGACTACAAGGCCGGCAAGCTGACCCACGAGGACAT MKLKIFHY GAAGCTGAAGATCTTCCACTAC 5 MKILELLINECGYKG 86 ATGAAGATCCTGGAGCTGCTGATCAACGAGTGCGGCTACAAGG LHLGGARKPDGIIYT GCCTGCACCTGGGCGGCGCCAGGAAGCCCGACGGCATCATCTAC EKEKYNYGVIIDTK ACCGAGAAGGAGAAGTACAACTACGGCGTGATCATCGACACCA AYSKGYNLPIGQIDE AGGCCTACAGCAAGGGCTACAACCTGCCCATCGGCCAGATCGAC MIRYIIENNERNIKR GAGATGATCAGGTACATCATCGAGAACAACGAGAGGAACATCA NTNCWWNNFEKNV AGAGGAACACCAACTGCTGGTGGAACAACTTCGAGAAGAACGT NEFYFSFISGEFTGNI GAACGAGTTCTACTTCAGCTTCATCAGCGGCGAGTTCACCGGCA EEKLNRIFISTNIKGN ACATCGAGGAGAAGCTGAACAGGATCTTCATCAGCACCAACATC AMSVKTLLYLANEI AAGGGCAACGCCATGAGCGTGAAGACCCTGCTGTACCTGGCCA KANRISYIELLNYFD ACGAGATCAAGGCCAACAGGATCAGCTACATCGAGCTGCTGAA NKV CTACTTCGACAACAAGGTG 6 AKSSQSETKEKLRE 87 GCCAAGAGCAGCCAGAGCGAGACCAAGGAGAAGCTGAGGGAG KLRNLPHEYLSLVD AAGCTGAGGAACCTGCCCCACGAGTACCTGAGCCTGGTGGACCT LAYDSKQNRLFEMK GGCCTACGACAGCAAGCAGAACAGGCTGTTCGAGATGAAGGTG VIELLTEECGFQGLH ATCGAGCTGCTGACCGAGGAGTGCGGCTTCCAGGGCCTGCACCT LGGSRRPDGVLYTA GGGCGGCAGCAGGAGGCCCGACGGCGTGCTGTACACCGCCGGC GLTDNYGIILDTKAY CTGACCGACAACTACGGCATCATCCTGGACACCAAGGCCTACAG SSGYSLPIAQADEME CAGCGGCTACAGCCTGCCCATCGCCCAGGCCGACGAGATGGAG RYVRENQTRDELVN AGGTACGTGAGGGAGAACCAGACCAGGGACGAGCTGGTGAACC PNQWWENFENGLG CCAACCAGTGGTGGGAGAACTTCGAGAACGGCCTGGGCACCTTC TFYFLFVAGHFNGN TACTTCCTGTTCGTGGCCGGCCACTTCAACGGCAACGTGCAGGC VQAQLERISRNTGV CCAGCTGGAGAGGATCAGCAGGAACACCGGCGTGCTGGGCGCC LGAAASISQLLLLAD GCCGCCAGCATCAGCCAGCTGCTGCTGCTGGCCGACGCCATCAG AIRGGRMDRERLRH GGGCGGCAGGATGGACAGGGAGAGGCTGAGGCACCTGATGTTC LMFQNEEFL CAGAACGAGGAGTTCCTG 7 NSEKSEFTQEKDNL 88 AACAGCGAGAAGAGCGAGTTCACCCAGGAGAAGGACAACCTGA REKLDTLSHEYLSLV GGGAGAAGCTGGACACCCTGAGCCACGAGTACCTGAGCCTGGT DLAFDSQQNRLFEM GGACCTGGCCTTCGACAGCCAGCAGAACAGGCTGTTCGAGATGA KTVELLTKECNYKG AGACCGTGGAGCTGCTGACCAAGGAGTGCAACTACAAGGGCGT VHLGGSRKPDGIIYT GCACCTGGGCGGCAGCAGGAAGCCCGACGGCATCATCTACACC ENSTDNYGVIIDTKA GAGAACAGCACCGACAACTACGGCGTGATCATCGACACCAAGG YSNGYNLPISQVDE CCTACAGCAACGGCTACAACCTGCCCATCAGCCAGGTGGACGAG MVRYVEENNKREK ATGGTGAGGTACGTGGAGGAGAACAACAAGAGGGAGAAGGAG ERNSNEWWKEFGD AGGAACAGCAACGAGTGGTGGAAGGAGTTCGGCGACAACATCA NINKFYFSFISGKFIG ACAAGTTCTACTTCAGCTTCATCAGCGGCAAGTTCATCGGCAAC NIEEKLQRITIFTNVY ATCGAGGAGAAGCTGCAGAGGATCACCATCTTCACCAACGTGTA GNAMTIITLLYLANE CGGCAACGCCATGACCATCATCACCCTGCTGTACCTGGCCAACG IKANRLKTMEVVKY AGATCAAGGCCAACAGGCTGAAGACCATGGAGGTGGTGAAGTA FDNKV CTTCGACAACAAGGTG 8 NLTCSDLTEIKEEVR 89 AACCTGACCTGCAGCGACCTGACCGAGATCAAGGAGGAGGTGA NALTHLSHEYLALID GGAACGCCCTGACCCACCTGAGCCACGAGTACCTGGCCCTGATC LAYDSTQNRLFEMK GACCTGGCCTACGACAGCACCCAGAACAGGCTGTTCGAGATGA TLQLLVEECGYQGT AGACCCTGCAGCTGCTGGTGGAGGAGTGCGGCTACCAGGGCAC HLGGSRKPDGICYSE CCACCTGGGCGGCAGCAGGAAGCCCGACGGCATCTGCTACAGC EAKSEGLEANYGIII GAGGAGGCCAAGAGCGAGGGCCTGGAGGCCAACTACGGCATCA DTKSYSGGYGLPISQ TCATCGACACCAAGAGCTACAGCGGCGGCTACGGCCTGCCCATC ADEMERYIRENQTR AGCCAGGCCGACGAGATGGAGAGGTACATCAGGGAGAACCAGA DAEVNRNKWWEAF CCAGGGACGCCGAGGTGAACAGGAACAAGTGGTGGGAGGCCTT PETIDIFYFMFVAGH CCCCGAGACCATCGACATCTTCTACTTCATGTTCGTGGCCGGCCA FKGNYFNQLERLQR CTTCAAGGGCAACTACTTCAACCAGCTGGAGAGGCTGCAGAGG STGIKGAAVDIKTLL AGCACCGGCATCAAGGGCGCCGCCGTGGACATCAAGACCCTGCT LTANRCKTGELDHA GCTGACCGCCAACAGGTGCAAGACCGGCGAGCTGGACCACGCC GIESCFFNNCRL GGCATCGAGAGCTGCTTCTTCAACAACTGCAGGCTG 9 DNVKSNFNQEKDEL 90 GACAACGTGAAGAGCAACTTCAACCAGGAGAAGGACGAGCTGA REKLDTLSHEYLYL GGGAGAAGCTGGACACCCTGAGCCACGAGTACCTGTACCTGCTG LDLAYDSKQNKLFE GACCTGGCCTACGACAGCAAGCAGAACAAGCTGTTCGAGATGA MKILELLINECGYRG AGATCCTGGAGCTGCTGATCAACGAGTGCGGCTACAGGGGCCTG LHLGGVRKPDGIIYT CACCTGGGCGGCGTGAGGAAGCCCGACGGCATCATCTACACCG EKEKYNYGVIIDTK AGAAGGAGAAGTACAACTACGGCGTGATCATCGACACCAAGGC AYSKGYNLPIGQIDE CTACAGCAAGGGCTACAACCTGCCCATCGGCCAGATCGACGAG MIRYIIENNERNIKR ATGATCAGGTACATCATCGAGAACAACGAGAGGAACATCAAGA NTNCWWNNFEKNV GGAACACCAACTGCTGGTGGAACAACTTCGAGAAGAACGTGAA NEFYFSFISGEFTGNI CGAGTTCTACTTCAGCTTCATCAGCGGCGAGTTCACCGGCAACA EEKLNRIFISTNIKGN TCGAGGAGAAGCTGAACAGGATCTTCATCAGCACCAACATCAA AMSVKTLLYLANEI GGGCAACGCCATGAGCGTGAAGACCCTGCTGTACCTGGCCAACG KANRISFLEMEKYF AGATCAAGGCCAACAGGATCAGCTTCCTGGAGATGGAGAAGTA DNKV CTTCGACAACAAGGTG 10 EGIKSNISLLKDELR 91 GAGGGCATCAAGAGCAACATCAGCCTGCTGAAGGACGAGCTGA GQISHISHEYLSLIDL GGGGCCAGATCAGCCACATCAGCCACGAGTACCTGAGCCTGATC AFDSKQNRLFEMKV GACCTGGCCTTCGACAGCAAGCAGAACAGGCTGTTCGAGATGA LELLVNEYGFKGRH AGGTGCTGGAGCTGCTGGTGAACGAGTACGGCTTCAAGGGCAG LGGSRKPDGIVYSTT GCACCTGGGCGGCAGCAGGAAGCCCGACGGCATCGTGTACAGC LEDNFGIIVDTKAYS ACCACCCTGGAGGACAACTTCGGCATCATCGTGGACACCAAGGC EGYSLPISQADEMER CTACAGCGAGGGCTACAGCCTGCCCATCAGCCAGGCCGACGAG YVRENSNRDEEVNP ATGGAGAGGTACGTGAGGGAGAACAGCAACAGGGACGAGGAG NKWWENFSEEVKK GTGAACCCCAACAAGTGGTGGGAGAACTTCAGCGAGGAGGTGA YYFVFISGSFKGKFE AGAAGTACTACTTCGTGTTCATCAGCGGCAGCTTCAAGGGCAAG EQLRRLSMTTGVNG TTCGAGGAGCAGCTGAGGAGGCTGAGCATGACCACCGGCGTGA SAVNVVNLLLGAEK ACGGCAGCGCCGTGAACGTGGTGAACCTGCTGCTGGGCGCCGA IRSGEMTIEELERAM GAAGATCAGGAGCGGCGAGATGACCATCGAGGAGCTGGAGAGG FNNSEFI GCCATGTTCAACAACAGCGAGTTCATC 11 ISKTNVLELKDKVR 92 ATCAGCAAGACCAACGTGCTGGAGCTGAAGGACAAGGTGAGGG DKLKYVDNRYLALI ACAAGCTGAAGTACGTGGACAACAGGTACCTGGCCCTGATCGAC DLAYDGTANRDFEI CTGGCCTACGACGGCACCGCCAACAGGGACTTCGAGATCCAGAC QTIDLLINELKFKGV CATCGACCTGCTGATCAACGAGCTGAAGTTCAAGGGCGTGAGGC RLGESRKPDGIISYDI TGGGCGAGAGCAGGAAGCCCGACGGCATCATCAGCTACGACAT NGVIIDNKAYSSGY CAACGGCGTGATCATCGACAACAAGGCCTACAGCAGCGGCTAC NLPINQADEMIRYIE AACCTGCCCATCAACCAGGCCGACGAGATGATCAGGTACATCGA ENQTRDKKINPNKW GGAGAACCAGACCAGGGACAAGAAGATCAACCCCAACAAGTGG WESFDDKVKDFNYL TGGGAGAGCTTCGACGACAAGGTGAAGGACTTCAACTACCTGTT FVSSFFKGNFKNNL CGTGAGCAGCTTCTTCAAGGGCAACTTCAAGAACAACCTGAAGC KHIANRTGVNGGVI ACATCGCCAACAGGACCGGCGTGAACGGCGGCGTGATCAACGT NVENLLYFAEELKS GGAGAACCTGCTGTACTTCGCCGAGGAGCTGAAGAGCGGCAGG GRLSYVDLFKMYDN CTGAGCTACGTGGACCTGTTCAAGATGTACGACAACGACGAGAT DEINI CAACATC 12 ISKTNVLELKDKVR 93 ATCAGCAAGACCAACGTGCTGGAGCTGAAGGACAAGGTGAGGG DKLKYVDHRYLALI ACAAGCTGAAGTACGTGGACCACAGGTACCTGGCCCTGATCGAC DLAYDGTANRDFEI CTGGCCTACGACGGCACCGCCAACAGGGACTTCGAGATCCAGAC QTIDLLINELKFKGV CATCGACCTGCTGATCAACGAGCTGAAGTTCAAGGGCGTGAGGC RLGESRKPDGIISYDI TGGGCGAGAGCAGGAAGCCCGACGGCATCATCAGCTACGACAT NGVIIDNKAYSTGY CAACGGCGTGATCATCGACAACAAGGCCTACAGCACCGGCTAC NLPINQADEMIRYIE AACCTGCCCATCAACCAGGCCGACGAGATGATCAGGTACATCGA ENQTRDKKINSNKW GGAGAACCAGACCAGGGACAAGAAGATCAACAGCAACAAGTGG WESFDDKVKNFNYL TGGGAGAGCTTCGACGACAAGGTGAAGAACTTCAACTACCTGTT FVSSFFKGNFKNNL CGTGAGCAGCTTCTTCAAGGGCAACTTCAAGAACAACCTGAAGC KHIANRTGVNGGAI ACATCGCCAACAGGACCGGCGTGAACGGCGGCGCCATCAACGT NVENLLYFAEELKA GGAGAACCTGCTGTACTTCGCCGAGGAGCTGAAGGCCGGCAGG GRLSYVDSFTMYDN CTGAGCTACGTGGACAGCTTCACCATGTACGACAACGACGAGAT DEIYV CTACGTG 13 KAEKSEFLIEKDKLR 94 AAGGCCGAGAAGAGCGAGTTCCTGATCGAGAAGGACAAGCTGA EKLDTLPHDYLSMV GGGAGAAGCTGGACACCCTGCCCCACGACTACCTGAGCATGGTG DLAYDSKQNRLFEM GACCTGGCCTACGACAGCAAGCAGAACAGGCTGTTCGAGATGA KTIELLINECNYKGL AGACCATCGAGCTGCTGATCAACGAGTGCAACTACAAGGGCCTG HLGGTRKPDGIVYT CACCTGGGCGGCACCAGGAAGCCCGACGGCATCGTGTACACCA NNEVENYGIIIDTKA ACAACGAGGTGGAGAACTACGGCATCATCATCGACACCAAGGC YSKGYNLPISQVDE CTACAGCAAGGGCTACAACCTGCCCATCAGCCAGGTGGACGAG MTRYVEENNKREK ATGACCAGGTACGTGGAGGAGAACAACAAGAGGGAGAAGAAG KRNPNEWWNNFDS AGGAACCCCAACGAGTGGTGGAACAACTTCGACAGCAACGTGA NVKKFYFSFISGKFV AGAAGTTCTACTTCAGCTTCATCAGCGGCAAGTTCGTGGGCAAC GNIEEKLQRITLFTEI ATCGAGGAGAAGCTGCAGAGGATCACCCTGTTCACCGAGATCTA YGNAITVTTLLYIAN CGGCAACGCCATCACCGTGACCACCCTGCTGTACATCGCCAACG EIKANRIVIKKSDIME AGATCAAGGCCAACAGGATGAAGAAGAGCGACATCATGGAGTA YFNDKV CTTCAACGACAAGGTG 14 ISKTNVLELKDKVR 95 ATCAGCAAGACCAACGTGCTGGAGCTGAAGGACAAGGTGAGGG DKLKYVDHRYLALI ACAAGCTGAAGTACGTGGACCACAGGTACCTGGCCCTGATCGAC DLAYDGTANRDFEI CTGGCCTACGACGGCACCGCCAACAGGGACTTCGAGATCCAGAC QTIDLLINELKFKGV CATCGACCTGCTGATCAACGAGCTGAAGTTCAAGGGCGTGAGGC RLGESRKPDGIISYNI TGGGCGAGAGCAGGAAGCCCGACGGCATCATCAGCTACAACAT NGVIIDNKAYSTGY CAACGGCGTGATCATCGACAACAAGGCCTACAGCACCGGCTAC NLPINQADEMIRYIE AACCTGCCCATCAACCAGGCCGACGAGATGATCAGGTACATCGA ENQTRDEKINSNKW GGAGAACCAGACCAGGGACGAGAAGATCAACAGCAACAAGTGG WESFDDEVKDFNYL TGGGAGAGCTTCGACGACGAGGTGAAGGACTTCAACTACCTGTT FVSSFFKGNFKNNL CGTGAGCAGCTTCTTCAAGGGCAACTTCAAGAACAACCTGAAGC KHIANRTGVNGGAI ACATCGCCAACAGGACCGGCGTGAACGGCGGCGCCATCAACGT NVENLLYFAEELKA GGAGAACCTGCTGTACTTCGCCGAGGAGCTGAAGGCCGGCAGG GRLSYVDSFTMYDN CTGAGCTACGTGGACAGCTTCACCATGTACGACAACGACGAGAT DEIYV CTACGTG 15 ISKTNILELKDKVRD 96 ATCAGCAAGACCAACATCCTGGAGCTGAAGGACAAGGTGAGGG KLKYVDHRYLALID ACAAGCTGAAGTACGTGGACCACAGGTACCTGGCCCTGATCGAC LAYDGTANRDFEIQ CTGGCCTACGACGGCACCGCCAACAGGGACTTCGAGATCCAGAC TIDLLINELKFKGVR CATCGACCTGCTGATCAACGAGCTGAAGTTCAAGGGCGTGAGGC LGESRKPDGIISYNIN TGGGCGAGAGCAGGAAGCCCGACGGCATCATCAGCTACAACAT GVIIDNKAYSTGYNL CAACGGCGTGATCATCGACAACAAGGCCTACAGCACCGGCTAC PINQADEMIRYIEEN AACCTGCCCATCAACCAGGCCGACGAGATGATCAGGTACATCGA QTRDEKINSNKWWE GGAGAACCAGACCAGGGACGAGAAGATCAACAGCAACAAGTGG SFDEKVKDFNYLFV TGGGAGAGCTTCGACGAGAAGGTGAAGGACTTCAACTACCTGTT SSFFKGNFKNNLKHI CGTGAGCAGCTTCTTCAAGGGCAACTTCAAGAACAACCTGAAGC ANRTGVNGGAINVE ACATCGCCAACAGGACCGGCGTGAACGGCGGCGCCATCAACGT NLLYFAEELKAGRIS GGAGAACCTGCTGTACTTCGCCGAGGAGCTGAAGGCCGGCAGG YLDSFKMYNNDEIY ATCAGCTACCTGGACAGCTTCAAGATGTACAACAACGACGAGAT L CTACCTG 16 ISKTNVLELKDKVR 97 ATCAGCAAGACCAACGTGCTGGAGCTGAAGGACAAGGTGAGGG DKLKYVDHRYLALI ACAAGCTGAAGTACGTGGACCACAGGTACCTGGCCCTGATCGAC DLAYDGTANRDFEI CTGGCCTACGACGGCACCGCCAACAGGGACTTCGAGATCCAGAC QTIDLLINELKFKGV CATCGACCTGCTGATCAACGAGCTGAAGTTCAAGGGCGTGAGGC RLGESRKPDGIISYNI TGGGCGAGAGCAGGAAGCCCGACGGCATCATCAGCTACAACAT NGVIIDNKAYSTGY CAACGGCGTGATCATCGACAACAAGGCCTACAGCACCGGCTAC NLPINQADEMIRYIE AACCTGCCCATCAACCAGGCCGACGAGATGATCAGGTACATCGA ENQTRDEKINSNKW GGAGAACCAGACCAGGGACGAGAAGATCAACAGCAACAAGTGG WESFDDKVKDFNYL TGGGAGAGCTTCGACGACAAGGTGAAGGACTTCAACTACCTGTT FVSSFFKGNFKNNL CGTGAGCAGCTTCTTCAAGGGCAACTTCAAGAACAACCTGAAGC KHIANRTGVSGGAI ACATCGCCAACAGGACCGGCGTGAGCGGCGGCGCCATCAACGT NVENLLYFAEELKA GGAGAACCTGCTGTACTTCGCCGAGGAGCTGAAGGCCGGCAGG GRLSYVDSFKMYDN CTGAGCTACGTGGACAGCTTCAAGATGTACGACAACGACGAGAT DEIYV CTACGTG 17 ISKTNVLELKDKVR 98 ATCAGCAAGACCAACGTGCTGGAGCTGAAGGACAAGGTGAGGA NKLKYVDHRYLALI ACAAGCTGAAGTACGTGGACCACAGGTACCTGGCCCTGATCGAC DLAYDGTANRDFEI CTGGCCTACGACGGCACCGCCAACAGGGACTTCGAGATCCAGAC QTIDLLINELKFKGV CATCGACCTGCTGATCAACGAGCTGAAGTTCAAGGGCGTGAGGC RLGESRKPDGIISYDI TGGGCGAGAGCAGGAAGCCCGACGGCATCATCAGCTACGACAT NGVIIDNKSYSTGYN CAACGGCGTGATCATCGACAACAAGAGCTACAGCACCGGCTAC LPINQADEMIRYIEE AACCTGCCCATCAACCAGGCCGACGAGATGATCAGGTACATCGA NQTRDEKINSNKW GGAGAACCAGACCAGGGACGAGAAGATCAACAGCAACAAGTGG WESFDEKVKDFNYL TGGGAGAGCTTCGACGAGAAGGTGAAGGACTTCAACTACCTGTT FVSSFFKGNFKNNL CGTGAGCAGCTTCTTCAAGGGCAACTTCAAGAACAACCTGAAGC KHIANRTGVNGGAI ACATCGCCAACAGGACCGGCGTGAACGGCGGCGCCATCAACGT NVENLLYFAEELKS GGAGAACCTGCTGTACTTCGCCGAGGAGCTGAAGAGCGGCAGG GRLSYVDSFTMYDN CTGAGCTACGTGGACAGCTTCACCATGTACGACAACGACGAGAT DEIYV CTACGTG 18 ISKTNVLELKDKVR 99 ATCAGCAAGACCAACGTGCTGGAGCTGAAGGACAAGGTGAGGG DKLKYVDHRYLSLI ACAAGCTGAAGTACGTGGACCACAGGTACCTGAGCCTGATCGAC DLAYDGNANRDFEI CTGGCCTACGACGGCAACGCCAACAGGGACTTCGAGATCCAGA QTIDLLINELNFKGV CCATCGACCTGCTGATCAACGAGCTGAACTTCAAGGGCGTGAGG RLGESRKPDGIISYNI CTGGGCGAGAGCAGGAAGCCCGACGGCATCATCAGCTACAACA NGVIIDNKAYSTGY TCAACGGCGTGATCATCGACAACAAGGCCTACAGCACCGGCTAC NLPINQADEMIRYIE AACCTGCCCATCAACCAGGCCGACGAGATGATCAGGTACATCGA ENQTRDEKINSNKW GGAGAACCAGACCAGGGACGAGAAGATCAACAGCAACAAGTGG WESFDDKVKDFNYL TGGGAGAGCTTCGACGACAAGGTGAAGGACTTCAACTACCTGTT FVSSFFKGNFKNNL CGTGAGCAGCTTCTTCAAGGGCAACTTCAAGAACAACCTGAAGC KHIANRTGVSGGAI ACATCGCCAACAGGACCGGCGTGAGCGGCGGCGCCATCAACGT NVENLLYFAEELKA GGAGAACCTGCTGTACTTCGCCGAGGAGCTGAAGGCCGGCAGG GRLSYADSFTMYDN CTGAGCTACGCCGACAGCTTCACCATGTACGACAACGACGAGAT DEIYV CTACGTG 19 IAKTNVLGLKDKVR 100 ATCGCCAAGACCAACGTGCTGGGCCTGAAGGACAAGGTGAGGG DRLKYVDHRYLALI ACAGGCTGAAGTACGTGGACCACAGGTACCTGGCCCTGATCGAC DLAYDGTANRDFEI CTGGCCTACGACGGCACCGCCAACAGGGACTTCGAGATCCAGAC QTIDLLINELKFKGV CATCGACCTGCTGATCAACGAGCTGAAGTTCAAGGGCGTGAGGC RLGESRKPDGIISYN TGGGCGAGAGCAGGAAGCCCGACGGCATCATCAGCTACAACGT VNGVIIDNKAYSKG GAACGGCGTGATCATCGACAACAAGGCCTACAGCAAGGGCTAC YNLPINQADEMIRYI AACCTGCCCATCAACCAGGCCGACGAGATGATCAGGTACATCGA EENQTRDEKINANK GGAGAACCAGACCAGGGACGAGAAGATCAACGCCAACAAGTGG WWESFDDKVEEFSY TGGGAGAGCTTCGACGACAAGGTGGAGGAGTTCAGCTACCTGTT LFVSSFFKGNFKNNL CGTGAGCAGCTTCTTCAAGGGCAACTTCAAGAACAACCTGAAGC KHIANRTGVNGGAI ACATCGCCAACAGGACCGGCGTGAACGGCGGCGCCATCAACGT NVENLLYFAEELKS GGAGAACCTGCTGTACTTCGCCGAGGAGCTGAAGAGCGGCAGG GRLSYMDSFSLYDN CTGAGCTACATGGACAGCTTCAGCCTGTACGACAACGACGAGAT DEICV CTGCGTG 20 ELKDEQSEKRKAKF 101 GAGCTGAAGGACGAGCAGAGCGAGAAGAGGAAGGCCAAGTTCC LKETKLPMKYIELLD TGAAGGAGACCAAGCTGCCCATGAAGTACATCGAGCTGCTGGA IAYDGKRNRDFEIVT CATCGCCTACGACGGCAAGAGGAACAGGGACTTCGAGATCGTG MELFREVYRLNSKL ACCATGGAGCTGTTCAGGGAGGTGTACAGGCTGAACAGCAAGC LGGGRKPDGLIYTD TGCTGGGCGGCGGCAGGAAGCCCGACGGCCTGATCTACACCGA DFGVIVDTKAYGEG CGACTTCGGCGTGATCGTGGACACCAAGGCCTACGGCGAGGGCT YSKSINQADEMIRYI ACAGCAAGAGCATCAACCAGGCCGACGAGATGATCAGGTACAT EDNKRRDEKRNPIK CGAGGACAACAAGAGGAGGGACGAGAAGAGGAACCCCATCAA WWESFPSSISQNNFY GTGGTGGGAGAGCTTCCCCAGCAGCATCAGCCAGAACAACTTCT FLWVSSKFVGKFQE ACTTCCTGTGGGTGAGCAGCAAGTTCGTGGGCAAGTTCCAGGAG QLAYTANETQTKGG CAGCTGGCCTACACCGCCAACGAGACCCAGACCAAGGGCGGCG AINVEQILIGADLIM CCATCAACGTGGAGCAGATCCTGATCGGCGCCGACCTGATCATG QKMLDINTIPSFFEN CAGAAGATGCTGGACATCAACACCATCCCCAGCTTCTTCGAGAA QEIIF CCAGGAGATCATCTTC 21 IFKTNVLELKDSIRE 102 ATCTTCAAGACCAACGTGCTGGAGCTGAAGGACAGCATCAGGG KLDYIDHRYLSLVD AGAAGCTGGACTACATCGACCACAGGTACCTGAGCCTGGTGGAC LAYDSKANRDFEIQ CTGGCCTACGACAGCAAGGCCAACAGGGACTTCGAGATCCAGA TIDLLINELDFKGLR CCATCGACCTGCTGATCAACGAGCTGGACTTCAAGGGCCTGAGG LGESRKPDGIISYDIN CTGGGCGAGAGCAGGAAGCCCGACGGCATCATCAGCTACGACA GVIIDNKAYSKGYN TCAACGGCGTGATCATCGACAACAAGGCCTACAGCAAGGGCTA LPINQADEMIRYIQE CAACCTGCCCATCAACCAGGCCGACGAGATGATCAGGTACATCC NQSRNEKINPNKWW AGGAGAACCAGAGCAGGAACGAGAAGATCAACCCCAACAAGTG ENFEDKVIKFNYLFI GTGGGAGAACTTCGAGGACAAGGTGATCAAGTTCAACTACCTGT SSLFVGGFKKNLQHI TCATCAGCAGCCTGTTCGTGGGCGGCTTCAAGAAGAACCTGCAG ANRTGVNGGAIDVE CACATCGCCAACAGGACCGGCGTGAACGGCGGCGCCATCGACG NLLYFAEEIKSGRLT TGGAGAACCTGCTGTACTTCGCCGAGGAGATCAAGAGCGGCAG YKDSFSRYINDEIKM GCTGACCTACAAGGACAGCTTCAGCAGGTACATCAACGACGAG ATCAAGATG 22 LPVKSEVSVFKDYL 103 CTGCCCGTGAAGAGCGAGGTGAGCGTGTTCAAGGACTACCTGAG RTHLTHVDHRYLIL GACCCACCTGACCCACGTGGACCACAGGTACCTGATCCTGGTGG VDLGFDGSSDRDYE ACCTGGGCTTCGACGGCAGCAGCGACAGGGACTACGAGATGAA MKTAELFTAELGFM GACCGCCGAGCTGTTCACCGCCGAGCTGGGCTTCATGGGCGCCA GARLGDTRKPDVCV GGCTGGGCGACACCAGGAAGCCCGACGTGTGCGTGTACCACGG YHGANGLIIDNKAY CGCCAACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGGC GKGYSLPIKQADEIY TACAGCCTGCCCATCAAGCAGGCCGACGAGATCTACAGGTACAT RYIEENKERDARLNP CGAGGAGAACAAGGAGAGGGACGCCAGGCTGAACCCCAACCAG NQWWKVFDESVTH TGGTGGAAGGTGTTCGACGAGAGCGTGACCCACTTCAGGTTCGC FRFAFISGSFTGGFK CTTCATCAGCGGCAGCTTCACCGGCGGCTTCAAGGACAGGATCG DRIELISMRSGICGA AGCTGATCAGCATGAGGAGCGGCATCTGCGGCGCCGCCGTGAA AVNSVNLLLMAEEL CAGCGTGAACCTGCTGCTGATGGCCGAGGAGCTGAAGAGCGGC KSGRLDYEEWFQYF AGGCTGGACTACGAGGAGTGGTTCCAGTACTTCGACTGCAACGA DCNDEISF CGAGATCAGCTTC 23 ISVKSDMAVVKDSV 104 ATCAGCGTGAAGAGCGACATGGCCGTGGTGAAGGACAGCGTGA RERLAHVSHEYLILI GGGAGAGGCTGGCCCACGTGAGCCACGAGTACCTGATCCTGATC DLGFDGTSDRDYEI GACCTGGGCTTCGACGGCACCAGCGACAGGGACTACGAGATCC QTAELFTRELDFLGG AGACCGCCGAGCTGTTCACCAGGGAGCTGGACTTCCTGGGCGGC RLGDTRKPDVCIYY AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCATCTACTACG GKDGMIIDNKAYGK GCAAGGACGGCATGATCATCGACAACAAGGCCTACGGCAAGGG GYSLPIKQADEMYR CTACAGCCTGCCCATCAAGCAGGCCGACGAGATGTACAGGTACC YLEENKERNEKINPN TGGAGGAGAACAAGGAGAGGAACGAGAAGATCAACCCCAACA RWWKVFDEGVTDY GGTGGTGGAAGGTGTTCGACGAGGGCGTGACCGACTACAGGTTC RFAFVSGSFTGGFKD GCCTTCGTGAGCGGCAGCTTCACCGGCGGCTTCAAGGACAGGCT RLENIHMRSGLCGG GGAGAACATCCACATGAGGAGCGGCCTGTGCGGCGGCGCCATC AIDSVTLLLLAEELK GACAGCGTGACCCTGCTGCTGCTGGCCGAGGAGCTGAAGGCCG AGRMEYSEFFRLFD GCAGGATGGAGTACAGCGAGTTCTTCAGGCTGTTCGACTGCAAC CNDEVTF GACGAGGTGACCTTC 24 ELKDKAADAVKAK 105 GAGCTGAAGGACAAGGCCGCCGACGCCGTGAAGGCCAAGTTCC FLKLTGLSMKYIELL TGAAGCTGACCGGCCTGAGCATGAAGTACATCGAGCTGCTGGAC DIAYDSSRNRDFEIL ATCGCCTACGACAGCAGCAGGAACAGGGACTTCGAGATCCTGA TADLFKNVYGLDA CCGCCGACCTGTTCAAGAACGTGTACGGCCTGGACGCCATGCAC MHLGGGRKPDAIAQ CTGGGCGGCGGCAGGAAGCCCGACGCCATCGCCCAGACCAGCC TSHFGIIIDTKAYGN ACTTCGGCATCATCATCGACACCAAGGCCTACGGCAACGGCTAC GYSKSISQEDEMVR AGCAAGAGCATCAGCCAGGAGGACGAGATGGTGAGGTACATCG YIEDNQQRSITRNSV AGGACAACCAGCAGAGGAGCATCACCAGGAACAGCGTGGAGTG EWWKNFNSSIPSTAF GTGGAAGAACTTCAACAGCAGCATCCCCAGCACCGCCTTCTACT YFLWVSSKFVGKFD TCCTGTGGGTGAGCAGCAAGTTCGTGGGCAAGTTCGACGACCAG DQLLATYNRTNTCG CTGCTGGCCACCTACAACAGGACCAACACCTGCGGCGGCGCCCT GALNVEQLLIGAYK GAACGTGGAGCAGCTGCTGATCGGCGCCTACAAGGTGAAGGCC VKAGLLGIGQIPSYF GGCCTGCTGGGCATCGGCCAGATCCCCAGCTACTTCAAGAACAA KNKEIAW GGAGATCGCCTGG 25 ISVKSDMAVVKDSV 106 ATCAGCGTGAAGAGCGACATGGCCGTGGTGAAGGACAGCGTGA RERLAHVSHEYLLLI GGGAGAGGCTGGCCCACGTGAGCCACGAGTACCTGCTGCTGATC DLGFDGTSDRDYEI GACCTGGGCTTCGACGGCACCAGCGACAGGGACTACGAGATCC QTAELLTRELDFLG AGACCGCCGAGCTGCTGACCAGGGAGCTGGACTTCCTGGGCGGC GRLGDTRKPDVCIY AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCATCTACTACG YGKDGMIIDNKAYG GCAAGGACGGCATGATCATCGACAACAAGGCCTACGGCAAGGG KGYSLPIKQADEMY CTACAGCCTGCCCATCAAGCAGGCCGACGAGATGTACAGGTACC RYLEENKERNEKINP TGGAGGAGAACAAGGAGAGGAACGAGAAGATCAACCCCAACA NRWWKVFDEGVTD GGTGGTGGAAGGTGTTCGACGAGGGCGTGACCGACTACAGGTTC YRFAFVSGSFTGGFK GCCTTCGTGAGCGGCAGCTTCACCGGCGGCTTCAAGGACAGGCT DRLENIHMRSGLCG GGAGAACATCCACATGAGGAGCGGCCTGTGCGGCGGCGCCATC GAIDSVTLLLLAEEL GACAGCGTGACCCTGCTGCTGCTGGCCGAGGAGCTGAAGGCCG KAGRMEYSEFFRLF GCAGGATGGAGTACAGCGAGTTCTTCAGGCTGTTCGACTGCAAC DCNDEVTF GACGAGGTGACCTTC 26 ELKDEQAEKRKAKF 107 GAGCTGAAGGACGAGCAGGCCGAGAAGAGGAAGGCCAAGTTCC LKETNLPMKYIELLD TGAAGGAGACCAACCTGCCCATGAAGTACATCGAGCTGCTGGAC IAYDGKRNRDFEIVT ATCGCCTACGACGGCAAGAGGAACAGGGACTTCGAGATCGTGA MELFRNVYRLHSKL CCATGGAGCTGTTCAGGAACGTGTACAGGCTGCACAGCAAGCTG LGGGRKPDGLLYQD CTGGGCGGCGGCAGGAAGCCCGACGGCCTGCTGTACCAGGACA RFGVIVDTKAYGKG GGTTCGGCGTGATCGTGGACACCAAGGCCTACGGCAAGGGCTAC YSKSINQADEMIRYI AGCAAGAGCATCAACCAGGCCGACGAGATGATCAGGTACATCG EDNKRRDENRNPIK AGGACAACAAGAGGAGGGACGAGAACAGGAACCCCATCAAGTG WWEAFPDTIPQEEF GTGGGAGGCCTTCCCCGACACCATCCCCCAGGAGGAGTTCTACT YFMWVSSKFIGKFQ TCATGTGGGTGAGCAGCAAGTTCATCGGCAAGTTCCAGGAGCAG EQLDYTSNETQIKG CTGGACTACACCAGCAACGAGACCCAGATCAAGGGCGCCGCCC AALNVEQLLLGADL TGAACGTGGAGCAGCTGCTGCTGGGCGCCGACCTGGTGCTGAAG VLKGQLHISDLPSYF GGCCAGCTGCACATCAGCGACCTGCCCAGCTACTTCCAGAACAA QNKEIEF GGAGATCGAGTTC 27 RNLDNVERDNRKAE 108 AGGAACCTGGACAACGTGGAGAGGGACAACAGGAAGGCCGAGT FLAKTSLPPRFIELLS TCCTGGCCAAGACCAGCCTGCCCCCCAGGTTCATCGAGCTGCTG IAYESKSNRDFEMIT AGCATCGCCTACGAGAGCAAGAGCAACAGGGACTTCGAGATGA AELFKDVYGLGAVH TCACCGCCGAGCTGTTCAAGGACGTGTACGGCCTGGGCGCCGTG LGNAKKPDALAFND CACCTGGGCAACGCCAAGAAGCCCGACGCCCTGGCCTTCAACGA DFGIIIDTKAYSNGY CGACTTCGGCATCATCATCGACACCAAGGCCTACAGCAACGGCT SKNINQEDEMVRYIE ACAGCAAGAACATCAACCAGGAGGACGAGATGGTGAGGTACAT DNQIRSPDRNNNEW CGAGGACAACCAGATCAGGAGCCCCGACAGGAACAACAACGAG WLSFPPSIPENDFHF TGGTGGCTGAGCTTCCCCCCCAGCATCCCCGAGAACGACTTCCA LWVSSYFTGRFEEQ CTTCCTGTGGGTGAGCAGCTACTTCACCGGCAGGTTCGAGGAGC LQETSARTGGTTGG AGCTGCAGGAGACCAGCGCCAGGACCGGCGGCACCACCGGCGG ALDVEQLLIGGSLIQ CGCCCTGGACGTGGAGCAGCTGCTGATCGGCGGCAGCCTGATCC EGSLAPHEVPAYMQ AGGAGGGCAGCCTGGCCCCCCACGAGGTGCCCGCCTACATGCA NRVIHF GAACAGGGTGATCCACTTC 28 SPVKSEVSVFKDYL 109 AGCCCCGTGAAGAGCGAGGTGAGCGTGTTCAAGGACTACCTGA RTHLTHVDHRYLIL GGACCCACCTGACCCACGTGGACCACAGGTACCTGATCCTGGTG VDLGFDGSSDRDYE GACCTGGGCTTCGACGGCAGCAGCGACAGGGACTACGAGATGA MKTAELFTAELGFM AGACCGCCGAGCTGTTCACCGCCGAGCTGGGCTTCATGGGCGCC GARLGDTRKPDVCV AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCGTGTACCACG YHGAHGLIIDNKAY GCGCCCACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGG GKGYSLPIKQADEIY CTACAGCCTGCCCATCAAGCAGGCCGACGAGATCTACAGGTACA RYIEENKERAVRLNP TCGAGGAGAACAAGGAGAGGGCCGTGAGGCTGAACCCCAACCA NQWWKVFDESVAH GTGGTGGAAGGTGTTCGACGAGAGCGTGGCCCACTTCAGGTTCG FRFAFISGSFTGGFK CCTTCATCAGCGGCAGCTTCACCGGCGGCTTCAAGGACAGGATC DRIELISMRSGICGA GAGCTGATCAGCATGAGGAGCGGCATCTGCGGCGCCGCCGTGA AVNSVNLLLMAEEL ACAGCGTGAACCTGCTGCTGATGGCCGAGGAGCTGAAGAGCGG KSGRLNYEEWFQYF CAGGCTGAACTACGAGGAGTGGTTCCAGTACTTCGACTGCAACG DCNDEISL ACGAGATCAGCCTG 29 TLVDIEKERKKAYFL 110 ACCCTGGTGGACATCGAGAAGGAGAGGAAGAAGGCCTACTTCC KETSLSPRYIELLEIA TGAAGGAGACCAGCCTGAGCCCCAGGTACATCGAGCTGCTGGA FDPKRNRDFE VITAE GATCGCCTTCGACCCCAAGAGGAACAGGGACTTCGAGGTGATCA LLKAGYGLKAKVLG CCGCCGAGCTGCTGAAGGCCGGCTACGGCCTGAAGGCCAAGGT GGRRPDGIAYTKDY GCTGGGCGGCGGCAGGAGGCCCGACGGCATCGCCTACACCAAG GLIVDTKAYSNGYG GACTACGGCCTGATCGTGGACACCAAGGCCTACAGCAACGGCTA KNIGQADEMIRYIED CGGCAAGAACATCGGCCAGGCCGACGAGATGATCAGGTACATC NQKRDNKRNPIEW GAGGACAACCAGAAGAGGGACAACAAGAGGAACCCCATCGAGT WREFEVQIPANSYY GGTGGAGGGAGTTCGAGGTGCAGATCCCCGCCAACAGCTACTAC YLWVSGRFTGRFDE TACCTGTGGGTGAGCGGCAGGTTCACCGGCAGGTTCGACGAGCA QLVYTSSQTNTRGG GCTGGTGTACACCAGCAGCCAGACCAACACCAGGGGCGGCGCC ALEVEQLLWGADA CTGGAGGTGGAGCAGCTGCTGTGGGGCGCCGACGCCGTGATGA VMKGKLNVSDLPK AGGGCAAGCTGAACGTGAGCGACCTGCCCAAGTACATGAACAA YMNNSIIKL CAGCATCATCAAGCTG 30 ELRDKVIEEQKAIFL 111 GAGCTGAGGGACAAGGTGATCGAGGAGCAGAAGGCCATCTTCC QKTKLPLSYIELLEIA TGCAGAAGACCAAGCTGCCCCTGAGCTACATCGAGCTGCTGGAG RDGKRSRDFELITIE ATCGCCAGGGACGGCAAGAGGAGCAGGGACTTCGAGCTGATCA LFKNIYKINARILGG CCATCGAGCTGTTCAAGAACATCTACAAGATCAACGCCAGGATC ARKPDGVLYMPEFG CTGGGCGGCGCCAGGAAGCCCGACGGCGTGCTGTACATGCCCG VIVDTKAYADGYSK AGTTCGGCGTGATCGTGGACACCAAGGCCTACGCCGACGGCTAC SIAQADEMIRYIEDN AGCAAGAGCATCGCCCAGGCCGACGAGATGATCAGGTACATCG KRRDPSRNSTKWWE AGGACAACAAGAGGAGGGACCCCAGCAGGAACAGCACCAAGTG HFPTSIPANNFYFLW GTGGGAGCACTTCCCCACCAGCATCCCCGCCAACAACTTCTACT VSSVFVNKFHEQLS TCCTGTGGGTGAGCAGCGTGTTCGTGAACAAGTTCCACGAGCAG YTAQETQTVGAALS CTGAGCTACACCGCCCAGGAGACCCAGACCGTGGGCGCCGCCCT VEQLLLGADSVLKG GAGCGTGGAGCAGCTGCTGCTGGGCGCCGACAGCGTGCTGAAG NLTTEKFIDSFKNQE GGCAACCTGACCACCGAGAAGTTCATCGACAGCTTCAAGAACCA IVF GGAGATCGTGTTC 31 GATKSDLSLLKDDIR 112 GGCGCCACCAAGAGCGACCTGAGCCTGCTGAAGGACGACATCA KKLNHINHKYLVLI GGAAGAAGCTGAACCACATCAACCACAAGTACCTGGTGCTGATC DLGFDGTADRDYEL GACCTGGGCTTCGACGGCACCGCCGACAGGGACTACGAGCTGC QTADLLTSELAFKG AGACCGCCGACCTGCTGACCAGCGAGCTGGCCTTCAAGGGCGCC ARLGDSRKPDVCVY AGGCTGGGCGACAGCAGGAAGCCCGACGTGTGCGTGTACCACG HDKNGLIIDNKAYG ACAAGAACGGCCTGATCATCGACAACAAGGCCTACGGCAGCGG SGYSLPIKQADEML CTACAGCCTGCCCATCAAGCAGGCCGACGAGATGCTGAGGTACA RYIEENQKRDKALN TCGAGGAGAACCAGAAGAGGGACAAGGCCCTGAACCCCAACGA PNEWWTIFDDAVSK GTGGTGGACCATCTTCGACGACGCCGTGAGCAAGTTCAACTTCG FNFAFVSGEFTGGFK CCTTCGTGAGCGGCGAGTTCACCGGCGGCTTCAAGGACAGGCTG DRLENISRRSYTNGA GAGAACATCAGCAGGAGGAGCTACACCAACGGCGCCGCCATCA AINSVNLLLLAEEIK ACAGCGTGAACCTGCTGCTGCTGGCCGAGGAGATCAAGAGCGG SGRISYGDAFTKFEC CAGGATCAGCTACGGCGACGCCTTCACCAAGTTCGAGTGCAACG NDEIII ACGAGATCATCATC 32 ELRNAALDKQKVNF 113 GAGCTGAGGAACGCCGCCCTGGACAAGCAGAAGGTGAACTTCA INKTGLPMKYIELLE TCAACAAGACCGGCCTGCCCATGAAGTACATCGAGCTGCTGGAG IAFDGSRNRDFEMV ATCGCCTTCGACGGCAGCAGGAACAGGGACTTCGAGATGGTGA TADLFKNVYGFNSIL CCGCCGACCTGTTCAAGAACGTGTACGGCTTCAACAGCATCCTG LGGGRKPDGLIFTDR CTGGGCGGCGGCAGGAAGCCCGACGGCCTGATCTTCACCGACA FGVIIDTKAYGNGYS GGTTCGGCGTGATCATCGACACCAAGGCCTACGGCAACGGCTAC KSIGQEDEMVRYIED AGCAAGAGCATCGGCCAGGAGGACGAGATGGTGAGGTACATCG NQLRDSNRNSVEW AGGACAACCAGCTGAGGGACAGCAACAGGAACAGCGTGGAGTG WKNFDEKIESENFYF GTGGAAGAACTTCGACGAGAAGATCGAGAGCGAGAACTTCTAC MWISSKFIGQFSDQL TTCATGTGGATCAGCAGCAAGTTCATCGGCCAGTTCAGCGACCA QSTSDRTNTKGAAL GCTGCAGAGCACCAGCGACAGGACCAACACCAAGGGCGCCGCC NVEQLLLGAAAARD CTGAACGTGGAGCAGCTGCTGCTGGGCGCCGCCGCCGCCAGGG GKLDINSLPIYMNNK ACGGCAAGCTGGACATCAACAGCCTGCCCATCTACATGAACAAC EILW AAGGAGATCCTGTGG 33 ELKDEQSEKRKAYF 114 GAGCTGAAGGACGAGCAGAGCGAGAAGAGGAAGGCCTACTTCC LKETNLPLKYIELLDI TGAAGGAGACCAACCTGCCCCTGAAGTACATCGAGCTGCTGGAC AYDGKRNRDFEIVT ATCGCCTACGACGGCAAGAGGAACAGGGACTTCGAGATCGTGA MELFRNVYRLQSKL CCATGGAGCTGTTCAGGAACGTGTACAGGCTGCAGAGCAAGCTG LGGVRKPDGLLYKH CTGGGCGGCGTGAGGAAGCCCGACGGCCTGCTGTACAAGCACA RFGIIVDTKAYGEGY GGTTCGGCATCATCGTGGACACCAAGGCCTACGGCGAGGGCTAC SKSISQADEMIRYIE AGCAAGAGCATCAGCCAGGCCGACGAGATGATCAGGTACATCG DNKRRDENRNSTK AGGACAACAAGAGGAGGGACGAGAACAGGAACAGCACCAAGT WWEHFPDCIPKQSF GGTGGGAGCACTTCCCCGACTGCATCCCCAAGCAGAGCTTCTAC YFMWVSSKFVGKFQ TTCATGTGGGTGAGCAGCAAGTTCGTGGGCAAGTTCCAGGAGCA EQLDYTANETKTNG GCTGGACTACACCGCCAACGAGACCAAGACCAACGGCGCCGCC AALNVEQLLWGAD CTGAACGTGGAGCAGCTGCTGTGGGGCGCCGACCTGGTGGCCAA LVAKGKLDISQLPSY GGGCAAGCTGGACATCAGCCAGCTGCCCAGCTACTTCCAGAACA FQNKEIEF AGGAGATCGAGTTC 34 HNNKFKNYLRENSE 115 CACAACAACAAGTTCAAGAACTACCTGAGGGAGAACAGCGAGC LSFKFIELIDIAYDGN TGAGCTTCAAGTTCATCGAGCTGATCGACATCGCCTACGACGGC RNRDMEIITAELLKE AACAGGAACAGGGACATGGAGATCATCACCGCCGAGCTGCTGA IYGLNVKLLGGGRK AGGAGATCTACGGCCTGAACGTGAAGCTGCTGGGCGGCGGCAG PDILAYTDDIGIIIDT GAAGCCCGACATCCTGGCCTACACCGACGACATCGGCATCATCA KAYKDGYGKQINQ TCGACACCAAGGCCTACAAGGACGGCTACGGCAAGCAGATCAA ADEMIRYIEDNQRR CCAGGCCGACGAGATGATCAGGTACATCGAGGACAACCAGAGG DLIRNPNEWWRYFP AGGGACCTGATCAGGAACCCCAACGAGTGGTGGAGGTACTTCCC KSISKEKIYFMWISS CAAGAGCATCAGCAAGGAGAAGATCTACTTCATGTGGATCAGC YFKNNFYEQVQYTA AGCTACTTCAAGAACAACTTCTACGAGCAGGTGCAGTACACCGC QETKSIGAALNVRQ CCAGGAGACCAAGAGCATCGGCGCCGCCCTGAACGTGAGGCAG LLLCADAIQKEVLSL CTGCTGCTGTGCGCCGACGCCATCCAGAAGGAGGTGCTGAGCCT DTFLGSFRNEEINL GGACACCTTCCTGGGCAGCTTCAGGAACGAGGAGATCAACCTG 35 LPVKSEVSILKDYLR 116 CTGCCCGTGAAGAGCGAGGTGAGCATCCTGAAGGACTACCTGA SHLTHIDHKYLILVD GGAGCCACCTGACCCACATCGACCACAAGTACCTGATCCTGGTG LGYDGTSDRDYEIQ GACCTGGGCTACGACGGCACCAGCGACAGGGACTACGAGATCC TAQLLTAELSFLGGR AGACCGCCCAGCTGCTGACCGCCGAGCTGAGCTTCCTGGGCGGC LGDTRKPDVCIYYE AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCATCTACTACG DNGLIIDNKAYGKG AGGACAACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGG YSLPMKQADEMYR CTACAGCCTGCCCATGAAGCAGGCCGACGAGATGTACAGGTAC YIEENKERSELLNPN ATCGAGGAGAACAAGGAGAGGAGCGAGCTGCTGAACCCCAACT CWWNIFDKDVKTFH GCTGGTGGAACATCTTCGACAAGGACGTGAAGACCTTCCACTTC FAFLSGEFTGGFRDR GCCTTCCTGAGCGGCGAGTTCACCGGCGGCTTCAGGGACAGGCT LNHISMRSGMRGAA GAACCACATCAGCATGAGGAGCGGCATGAGGGGCGCCGCCGTG VNSANLLIMAEKLK AACAGCGCCAACCTGCTGATCATGGCCGAGAAGCTGAAGGCCG AGTMEYEEFFRLFD GCACCATGGAGTACGAGGAGTTCTTCAGGCTGTTCGACACCAAC TNDEILF GACGAGATCCTGTTC 36 LPVKSQVSILKDYLR 117 CTGCCCGTGAAGAGCCAGGTGAGCATCCTGAAGGACTACCTGAG SYLSHVDHKYLILLD GAGCTACCTGAGCCACGTGGACCACAAGTACCTGATCCTGCTGG LGFDGTSDRDYEIW ACCTGGGCTTCGACGGCACCAGCGACAGGGACTACGAGATCTG TAQLLTAELSFLGGR GACCGCCCAGCTGCTGACCGCCGAGCTGAGCTTCCTGGGCGGCA LGDTRKPDVCIYYE GGCTGGGCGACACCAGGAAGCCCGACGTGTGCATCTACTACGA DNGLIIDNKAYGKG GGACAACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGGC YSLPIKQADEMYRYI TACAGCCTGCCCATCAAGCAGGCCGACGAGATGTACAGGTACAT EENKERSDLLNPNC CGAGGAGAACAAGGAGAGGAGCGACCTGCTGAACCCCAACTGC WWNIFGEGVKTFRF TGGTGGAACATCTTCGGCGAGGGCGTGAAGACCTTCAGGTTCGC AFLSGEFTGGFKDRL CTTCCTGAGCGGCGAGTTCACCGGCGGCTTCAAGGACAGGCTGA NHISMRSGIKGAAV ACCACATCAGCATGAGGAGCGGCATCAAGGGCGCCGCCGTGAA NSANLLIMAEQLKS CAGCGCCAACCTGCTGATCATGGCCGAGCAGCTGAAGAGCGGC GTMSYEEFFQLFDY ACCATGAGCTACGAGGAGTTCTTCCAGCTGTTCGACTACAACGA NDEIIF CGAGATCATCTTC 37 VSKTNILELKDNTRE 118 GTGAGCAAGACCAACATCCTGGAGCTGAAGGACAACACCAGGG KLVYLDHRYLSLFD AGAAGCTGGTGTACCTGGACCACAGGTACCTGAGCCTGTTCGAC LAYDDKASRDFEIQ CTGGCCTACGACGACAAGGCCAGCAGGGACTTCGAGATCCAGA TIDLLINELQFKGLR CCATCGACCTGCTGATCAACGAGCTGCAGTTCAAGGGCCTGAGG LGERRKPDGIISYGV CTGGGCGAGAGGAGGAAGCCCGACGGCATCATCAGCTACGGCG NGVIIDNKAYSKGY TGAACGGCGTGATCATCGACAACAAGGCCTACAGCAAGGGCTA NLPIRQADEMIRYIQ CAACCTGCCCATCAGGCAGGCCGACGAGATGATCAGGTACATCC ENQSRDEKLNPNKW AGGAGAACCAGAGCAGGGACGAGAAGCTGAACCCCAACAAGTG WENFEEETSKFNYL GTGGGAGAACTTCGAGGAGGAGACCAGCAAGTTCAACTACCTG FISSKFISGFKKNLQY TTCATCAGCAGCAAGTTCATCAGCGGCTTCAAGAAGAACCTGCA IADRTGVNGGAINV GTACATCGCCGACAGGACCGGCGTGAACGGCGGCGCCATCAAC ENLLCFAEMLKSGK GTGGAGAACCTGCTGTGCTTCGCCGAGATGCTGAAGAGCGGCAA LEYNDFFNQYNNDE GCTGGAGTACAACGACTTCTTCAACCAGTACAACAACGACGAGA IIM TCATCATG 38 LPVKSQVSILKDYLR 119 CTGCCCGTGAAGAGCCAGGTGAGCATCCTGAAGGACTACCTGAG SCLSHVDHKYLILLD GAGCTGCCTGAGCCACGTGGACCACAAGTACCTGATCCTGCTGG LGFDGTSDRDYEIQT ACCTGGGCTTCGACGGCACCAGCGACAGGGACTACGAGATCCA AQLLTAELSFLGGRL GACCGCCCAGCTGCTGACCGCCGAGCTGAGCTTCCTGGGCGGCA GDTRKPDVCIYYED GGCTGGGCGACACCAGGAAGCCCGACGTGTGCATCTACTACGA NGLIIDNKAYGKGY GGACAACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGGC SLPIKQADEMYRYIE TACAGCCTGCCCATCAAGCAGGCCGACGAGATGTACAGGTACAT ENKERSELLNPNCW CGAGGAGAACAAGGAGAGGAGCGAGCTGCTGAACCCCAACTGC WNIFDEGVKTFRFA TGGTGGAACATCTTCGACGAGGGCGTGAAGACCTTCAGGTTCGC FLSGEFTGGFKDRLN CTTCCTGAGCGGCGAGTTCACCGGCGGCTTCAAGGACAGGCTGA HISMRSGIKGAAVNS ACCACATCAGCATGAGGAGCGGCATCAAGGGCGCCGCCGTGAA ANLLIIAEQLKSGTM CAGCGCCAACCTGCTGATCATCGCCGAGCAGCTGAAGAGCGGC SYEEFFQLFDQNDEI ACCATGAGCTACGAGGAGTTCTTCCAGCTGTTCGACCAGAACGA TV CGAGATCACCGTG 39 MSSKSEISVIKDNIR 120 ATGAGCAGCAAGAGCGAGATCAGCGTGATCAAGGACAACATCA KRLNHINHKYLVLID GGAAGAGGCTGAACCACATCAACCACAAGTACCTGGTGCTGATC LGFDGTADRDYELQ GACCTGGGCTTCGACGGCACCGCCGACAGGGACTACGAGCTGC TADLLTSELSFKGAR AGACCGCCGACCTGCTGACCAGCGAGCTGAGCTTCAAGGGCGCC LGDTRKPDVCVYHG AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCGTGTACCACG TNGLIIDNKAYGKG GCACCAACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGG YSLPIKQADEMLRYI CTACAGCCTGCCCATCAAGCAGGCCGACGAGATGCTGAGGTACA EENQKRDKSLNPNE TCGAGGAGAACCAGAAGAGGGACAAGAGCCTGAACCCCAACGA WWTIFDDAVSKFNF GTGGTGGACCATCTTCGACGACGCCGTGAGCAAGTTCAACTTCG AFVSGEFTGGFKDR CCTTCGTGAGCGGCGAGTTCACCGGCGGCTTCAAGGACAGGCTG LENISRRSSVNGAAI GAGAACATCAGCAGGAGGAGCAGCGTGAACGGCGCCGCCATCA NSVNLLLLAEEIKSG ACAGCGTGAACCTGCTGCTGCTGGCCGAGGAGATCAAGAGCGG RMSYSDAFKNFDCN CAGGATGAGCTACAGCGACGCCTTCAAGAACTTCGACTGCAACA KEITI AGGAGATCACCATC 40 RNLDKVERDSRKAE 121 AGGAACCTGGACAAGGTGGAGAGGGACAGCAGGAAGGCCGAGT FLAKTSLPPRFIELLS TCCTGGCCAAGACCAGCCTGCCCCCCAGGTTCATCGAGCTGCTG IAYESKSNRDFEMIT AGCATCGCCTACGAGAGCAAGAGCAACAGGGACTTCGAGATGA AEFFKDVYGLGAVH TCACCGCCGAGTTCTTCAAGGACGTGTACGGCCTGGGCGCCGTG LGNARKPDALAFTD CACCTGGGCAACGCCAGGAAGCCCGACGCCCTGGCCTTCACCGA NFGIVIDTKAYSNGY CAACTTCGGCATCGTGATCGACACCAAGGCCTACAGCAACGGCT SKNINQEDEMVRYIE ACAGCAAGAACATCAACCAGGAGGACGAGATGGTGAGGTACAT DNQIRSPERNKNEW CGAGGACAACCAGATCAGGAGCCCCGAGAGGAACAAGAACGAG WLSFPPSIPENNFHF TGGTGGCTGAGCTTCCCCCCCAGCATCCCCGAGAACAACTTCCA LWVSSYFTGYFEEQ CTTCCTGTGGGTGAGCAGCTACTTCACCGGCTACTTCGAGGAGC LQETSDRAGGMTGG AGCTGCAGGAGACCAGCGACAGGGCCGGCGGCATGACCGGCGG ALDIEQLLIGGSLVQ CGCCCTGGACATCGAGCAGCTGCTGATCGGCGGCAGCCTGGTGC EGKLAPHDIPEYMQ AGGAGGGCAAGCTGGCCCCCCACGACATCCCCGAGTACATGCA NRVIHF GAACAGGGTGATCCACTTC 41 APVKSEVSLCKDILR 122 GCCCCCGTGAAGAGCGAGGTGAGCCTGTGCAAGGACATCCTGA SHLTHVDHKYLILL GGAGCCACCTGACCCACGTGGACCACAAGTACCTGATCCTGCTG DLGFDGTSDRDYEI GACCTGGGCTTCGACGGCACCAGCGACAGGGACTACGAGATCC QTAQLLTAELDFKG AGACCGCCCAGCTGCTGACCGCCGAGCTGGACTTCAAGGGCGCC ARLGDTRKPDVCVY AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCGTGTACTACG YGEDGLILDNKAYG GCGAGGACGGCCTGATCCTGGACAACAAGGCCTACGGCAAGGG KGYSLPIKQADEMY CTACAGCCTGCCCATCAAGCAGGCCGACGAGATGTACAGGTACA RYIEENKERNERLNP TCGAGGAGAACAAGGAGAGGAACGAGAGGCTGAACCCCAACAA NKWWEIFDKDVVR GTGGTGGGAGATCTTCGACAAGGACGTGGTGAGGTACCACTTCG YHFAFVSGTFTGGF CCTTCGTGAGCGGCACCTTCACCGGCGGCTTCAAGGAGAGGCTG KERLDNIRMRSGICG GACAACATCAGGATGAGGAGCGGCATCTGCGGCGCCGCCGTGA AAVNSMNLLLMAE ACAGCATGAACCTGCTGCTGATGGCCGAGGAGCTGAAGAGCGG ELKSGRLGYKECFA CAGGCTGGGCTACAAGGAGTGCTTCGCCCTGTTCGACTGCAACG LFDCNDEIAF ACGAGATCGCCTTC 42 SCVKDEVNDIVDRV 123 AGCTGCGTGAAGGACGAGGTGAACGACATCGTGGACAGGGTGA RVKLKNIDHKYLILI GGGTGAAGCTGAAGAACATCGACCACAAGTACCTGATCCTGATC SLAYSDETERTKKN AGCCTGGCCTACAGCGACGAGACCGAGAGGACCAAGAAGAACA SDARDFEIQTAELFT GCGACGCCAGGGACTTCGAGATCCAGACCGCCGAGCTGTTCACC KELGFNGIRLGESNK AAGGAGCTGGGCTTCAACGGCATCAGGCTGGGCGAGAGCAACA PDVLISFGANGTIIDN AGCCCGACGTGCTGATCAGCTTCGGCGCCAACGGCACCATCATC KSYKDGFNIPRVTSD GACAACAAGAGCTACAAGGACGGCTTCAACATCCCCAGGGTGA QMIRYINENNQRTT CCAGCGACCAGATGATCAGGTACATCAACGAGAACAACCAGAG QLNPNEWWKNFDSS GACCACCCAGCTGAACCCCAACGAGTGGTGGAAGAACTTCGAC VSNYTFLFVTSFLKG AGCAGCGTGAGCAACTACACCTTCCTGTTCGTGACCAGCTTCCT SFKNQIEYISNATNG GAAGGGCAGCTTCAAGAACCAGATCGAGTACATCAGCAACGCC TRGAAINVESLLYIS ACCAACGGCACCAGGGGCGCCGCCATCAACGTGGAGAGCCTGC EDIKSGKIKQSDFYS TGTACATCAGCGAGGACATCAAGAGCGGCAAGATCAAGCAGAG EFKNDEIVY CGACTTCTACAGCGAGTTCAAGAACGACGAGATCGTGTAC 43 SQGDKAREQLKAKF 124 AGCCAGGGCGACAAGGCCAGGGAGCAGCTGAAGGCCAAGTTCC LAKTNLLPRYVELL TGGCCAAGACCAACCTGCTGCCCAGGTACGTGGAGCTGCTGGAC DIAYDSKRNRDFEM ATCGCCTACGACAGCAAGAGGAACAGGGACTTCGAGATGGTGA VTAELFNFAYLLPA CCGCCGAGCTGTTCAACTTCGCCTACCTGCTGCCCGCCGTGCACC VHLGGVRKPDALVA TGGGCGGCGTGAGGAAGCCCGACGCCCTGGTGGCCACCAAGAA TKKFGIIVDTKAYAN GTTCGGCATCATCGTGGACACCAAGGCCTACGCCAACGGCTACA GYSRNANQADEMA GCAGGAACGCCAACCAGGCCGACGAGATGGCCAGGTACATCAC RYITENQKRDPKTNP CGAGAACCAGAAGAGGGACCCCAAGACCAACCCCAACAGGTGG NRWWDNFDARIPPN TGGGACAACTTCGACGCCAGGATCCCCCCCAACGCCTACTACTT AYYFLWVSSFFTGQ CCTGTGGGTGAGCAGCTTCTTCACCGGCCAGTTCGACGACCAGC FDDQLSYTAHRTNT TGAGCTACACCGCCCACAGGACCAACACCCACGGCGGCGCCCTG HGGALNVEQLLIGA AACGTGGAGCAGCTGCTGATCGGCGCCAACATGATCCAGACCG NMIQTGQLDRNKLP GCCAGCTGGACAGGAACAAGCTGCCCGAGTACATGCAGGACAA EYMQDKEITF GGAGATCACCTTC 44 KVQKSNILDVIEKCR 125 AAGGTGCAGAAGAGCAACATCCTGGACGTGATCGAGAAGTGCA EKINNIPHEYLALIP GGGAGAAGATCAACAACATCCCCCACGAGTACCTGGCCCTGATC MSFDENESTMFEIKT CCCATGAGCTTCGACGAGAACGAGAGCACCATGTTCGAGATCAA IELLTEHCKFDGLHC GACCATCGAGCTGCTGACCGAGCACTGCAAGTTCGACGGCCTGC GGASKPDGLIYSED ACTGCGGCGGCGCCAGCAAGCCCGACGGCCTGATCTACAGCGA YGVIIDTKSYKDGFN GGACTACGGCGTGATCATCGACACCAAGAGCTACAAGGACGGC IQTPERDKMKRYIEE TTCAACATCCAGACCCCCGAGAGGGACAAGATGAAGAGGTACA NQNRNPQHNKTRW TCGAGGAGAACCAGAACAGGAACCCCCAGCACAACAAGACCAG WDEFPHNISNFLFLF GTGGTGGGACGAGTTCCCCCACAACATCAGCAACTTCCTGTTCC VSGKFGGNFKEQLRI TGTTCGTGAGCGGCAAGTTCGGCGGCAACTTCAAGGAGCAGCTG LSEQTNNTLGGALSS AGGATCCTGAGCGAGCAGACCAACAACACCCTGGGCGGCGCCC YVLLNIAEQIAINKID TGAGCAGCTACGTGCTGCTGAACATCGCCGAGCAGATCGCCATC HCDFKTRISCLDEVA AACAAGATCGACCACTGCGACTTCAAGACCAGGATCAGCTGCCT GGACGAGGTGGCC 45 VPVKSEVSLCKDYL 126 GTGCCCGTGAAGAGCGAGGTGAGCCTGTGCAAGGACTACCTGA RSYLTHVDHKYLILL GGAGCTACCTGACCCACGTGGACCACAAGTACCTGATCCTGCTG DLGFDGTSDRDYEI GACCTGGGCTTCGACGGCACCAGCGACAGGGACTACGAGATCC QTAQLLTAELDFKG AGACCGCCCAGCTGCTGACCGCCGAGCTGGACTTCAAGGGCGCC ARLGDTRKPDVCVY AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCGTGTACTACG YGEDGLIIDNKAYG GCGAGGACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGG KGYSLPIKQADEIYR CTACAGCCTGCCCATCAAGCAGGCCGACGAGATCTACAGGTACA YIEENKKRDEKLNP TCGAGGAGAACAAGAAGAGGGACGAGAAGCTGAACCCCAACAA NKWWEIFDKGVVR GTGGTGGGAGATCTTCGACAAGGGCGTGGTGAGGTACCACTTCG YHFAFVSGAFTGGF CCTTCGTGAGCGGCGCCTTCACCGGCGGCTTCAAGGAGAGGCTG KERLDNIRMRSGICG GACAACATCAGGATGAGGAGCGGCATCTGCGGCGCCGCCATCA AAINSMNLLLMAEE ACAGCATGAACCTGCTGCTGATGGCCGAGGAGCTGAAGAGCGG LKSGRLGYEECFALF CAGGCTGGGCTACGAGGAGTGCTTCGCCCTGTTCGACTGCAACG DCNDEITF ACGAGATCACCTTC 46 VPVKSEVSLCKDYL 127 GTGCCCGTGAAGAGCGAGGTGAGCCTGTGCAAGGACTACCTGA RSHLNHVDHRYLIL GGAGCCACCTGAACCACGTGGACCACAGGTACCTGATCCTGCTG LDLGFDGTSDRDYEI GACCTGGGCTTCGACGGCACCAGCGACAGGGACTACGAGATCC QTAQLLTGELNFKG AGACCGCCCAGCTGCTGACCGGCGAGCTGAACTTCAAGGGCGCC ARLGDTRKPDVCVY AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCGTGTACTACG YGEDGLIIDNKAYG GCGAGGACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGG KGYSLPIKQADEMY CTACAGCCTGCCCATCAAGCAGGCCGACGAGATGTACAGGTACA RYIEENKERNEKLNP TCGAGGAGAACAAGGAGAGGAACGAGAAGCTGAACCCCAACAA NKWWEIFDKDVIHY GTGGTGGGAGATCTTCGACAAGGACGTGATCCACTACCACTTCG HFAFVSGAFTGGFK CCTTCGTGAGCGGCGCCTTCACCGGCGGCTTCAAGGAGAGGCTG ERLENIRMRSGIYGA GAGAACATCAGGATGAGGAGCGGCATCTACGGCGCCGCCGTGA AVNSMNLLLMAEEL ACAGCATGAACCTGCTGCTGATGGCCGAGGAGCTGAAGAGCGG KSGRLDYKECFKLF CAGGCTGGACTACAAGGAGTGCTTCAAGCTGTTCGACTGCAACG DCNDEIVL ACGAGATCGTGCTG 47 VPVKSEVSLLKDYL 128 GTGCCCGTGAAGAGCGAGGTGAGCCTGCTGAAGGACTACCTGA RSHLVHVDHKYLVL GGAGCCACCTGGTGCACGTGGACCACAAGTACCTGGTGCTGCTG LDLGFDGTSDRDYEI GACCTGGGCTTCGACGGCACCAGCGACAGGGACTACGAGATCC QTAQLLTGELNFKG AGACCGCCCAGCTGCTGACCGGCGAGCTGAACTTCAAGGGCGCC ARLGDTRKPDVCVY AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCGTGTACTACG YGEDGLIIDNKAYG GCGAGGACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGG KGYSLPIKQADEMY CTACAGCCTGCCCATCAAGCAGGCCGACGAGATGTACAGGTACA RYIEENKERNEKLNP TCGAGGAGAACAAGGAGAGGAACGAGAAGCTGAACCCCAACAA NKWWEIFGNDVIHY GTGGTGGGAGATCTTCGGCAACGACGTGATCCACTACCACTTCG HFAFVSGAFTGGFK CCTTCGTGAGCGGCGCCTTCACCGGCGGCTTCAAGGAGAGGCTG ERLDNIRMRSGIYGA GACAACATCAGGATGAGGAGCGGCATCTACGGCGCCGCCGTGA AVNSMNLLLLAEEL ACAGCATGAACCTGCTGCTGCTGGCCGAGGAGCTGAAGAGCGG KSGRLGYKECFKLF CAGGCTGGGCTACAAGGAGTGCTTCAAGCTGTTCGACTGCAACG DCNDEIVL ACGAGATCGTGCTG 48 ECVKDNVVDIKDRV 129 GAGTGCGTGAAGGACAACGTGGTGGACATCAAGGACAGGGTGA RNKLIHLDHKYLALI GGAACAAGCTGATCCACCTGGACCACAAGTACCTGGCCCTGATC DLAYSDAASRAKKN GACCTGGCCTACAGCGACGCCGCCAGCAGGGCCAAGAAGAACG ADAREFEIQTADLFT CCGACGCCAGGGAGTTCGAGATCCAGACCGCCGACCTGTTCACC KELSFNGQRLGDSR AAGGAGCTGAGCTTCAACGGCCAGAGGCTGGGCGACAGCAGGA KPDVIISYGLDGTIV AGCCCGACGTGATCATCAGCTACGGCCTGGACGGCACCATCGTG DNKSYKDGFNISRT GACAACAAGAGCTACAAGGACGGCTTCAACATCAGCAGGACCT CADEMSRYINENNL GCGCCGACGAGATGAGCAGGTACATCAACGAGAACAACCTGAG RQKSLNPNEWWKN GCAGAAGAGCCTGAACCCCAACGAGTGGTGGAAGAACTTCGAC FDSTITAYTFLFITSY AGCACCATCACCGCCTACACCTTCCTGTTCATCACCAGCTACCTG LKGQFEDQLEYVSN AAGGGCCAGTTCGAGGACCAGCTGGAGTACGTGAGCAACGCCA ANGGIKGAAIGVESL ACGGCGGCATCAAGGGCGCCGCCATCGGCGTGGAGAGCCTGCT LYLSEGIKAGRISHA GTACCTGAGCGAGGGCATCAAGGCCGGCAGGATCAGCCACGCC DFYSNFNNKEMIY GACTTCTACAGCAACTTCAACAACAAGGAGATGATCTAC 49 IAKSDFSIIKDNIRRK 130 ATCGCCAAGAGCGACTTCAGCATCATCAAGGACAACATCAGGA LQYVNHKYLLLIDL GGAAGCTGCAGTACGTGAACCACAAGTACCTGCTGCTGATCGAC GFDSDSNRDYEIQTA CTGGGCTTCGACAGCGACAGCAACAGGGACTACGAGATCCAGA ELLTTELAFKGARL CCGCCGAGCTGCTGACCACCGAGCTGGCCTTCAAGGGCGCCAGG GDTRKPDVCVYYGE CTGGGCGACACCAGGAAGCCCGACGTGTGCGTGTACTACGGCG NGLIIDNKAYSKGYS AGAACGGCCTGATCATCGACAACAAGGCCTACAGCAAGGGCTA LPMSQADEMVRYIE CAGCCTGCCCATGAGCCAGGCCGACGAGATGGTGAGGTACATC ENKARQSSINPNQW GAGGAGAACAAGGCCAGGCAGAGCAGCATCAACCCCAACCAGT WKIFEDTVCNFNYA GGTGGAAGATCTTCGAGGACACCGTGTGCAACTTCAACTACGCC FVSGEFTGGFKDRL TTCGTGAGCGGCGAGTTCACCGGCGGCTTCAAGGACAGGCTGAA NNICERTRVSGGAIN CAACATCTGCGAGAGGACCAGGGTGAGCGGCGGCGCCATCAAC TINLLLLAEELKSGR ACCATCAACCTGCTGCTGCTGGCCGAGGAGCTGAAGAGCGGCA MSYPKCFSYFDTND GGATGAGCTACCCCAAGTGCTTCAGCTACTTCGACACCAACGAC EVHI GAGGTGCACATC 50 LKYLGIKKQNRAFEI 131 CTGAAGTACCTGGGCATCAAGAAGCAGAACAGGGCCTTCGAGA ITAELFNTSYKLSAT TCATCACCGCCGAGCTGTTCAACACCAGCTACAAGCTGAGCGCC HLGGGRRPDVLVYN ACCCACCTGGGCGGCGGCAGGAGGCCCGACGTGCTGGTGTACA DNFGIIVDTKAYKD ACGACAACTTCGGCATCATCGTGGACACCAAGGCCTACAAGGAC GYGRNVNQEDEMV GGCTACGGCAGGAACGTGAACCAGGAGGACGAGATGGTGAGGT RYITENNIRKQDINK ACATCACCGAGAACAACATCAGGAAGCAGGACATCAACAAGAA NDWWKYFSKSIPST CGACTGGTGGAAGTACTTCAGCAAGAGCATCCCCAGCACCAGCT SYYHLWISSQFVGM ACTACCACCTGTGGATCAGCAGCCAGTTCGTGGGCATGTTCAGC FSDQLRETSSRTGEN GACCAGCTGAGGGAGACCAGCAGCAGGACCGGCGAGAACGGCG GGAMNVEQLLIGAN GCGCCATGAACGTGGAGCAGCTGCTGATCGGCGCCAACCAGGT QVLNNVLDPNCLPK GCTGAACAACGTGCTGGACCCCAACTGCCTGCCCAAGTACATGG YMENKEIIF AGAACAAGGAGATCATCTTC 51 VPVKSEVSLCKDYL 132 GTGCCCGTGAAGAGCGAGGTGAGCCTGTGCAAGGACTACCTGA RSHLNHVDHKYLIL GGAGCCACCTGAACCACGTGGACCACAAGTACCTGATCCTGCTG LDLGFDGTSDRDYEI GACCTGGGCTTCGACGGCACCAGCGACAGGGACTACGAGATCC QTAQLLTGELNFKG AGACCGCCCAGCTGCTGACCGGCGAGCTGAACTTCAAGGGCGCC ARLGDTRKPDVCVY AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCGTGTACTACG YGEDGLIIDNKAYG GCGAGGACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGG KGYSLPIKQADEMY CTACAGCCTGCCCATCAAGCAGGCCGACGAGATGTACAGGTACA RYIEENKERNEKLNP TCGAGGAGAACAAGGAGAGGAACGAGAAGCTGAACCCCAACAA NKWWEIFDKDVIHY GTGGTGGGAGATCTTCGACAAGGACGTGATCCACTACCACTTCG HFAFVSGAFTGGFR CCTTCGTGAGCGGCGCCTTCACCGGCGGCTTCAGGGAGAGGCTG ERLENIRMRSGIYGA GAGAACATCAGGATGAGGAGCGGCATCTACGGCGCCGCCGTGA AVNSMNLLLMAEEL ACAGCATGAACCTGCTGCTGATGGCCGAGGAGCTGAAGAGCGG KSGRLGYKECFKLF CAGGCTGGGCTACAAGGAGTGCTTCAAGCTGTTCGACTGCAACG DCNDEIVL ACGAGATCGTGCTG 52 VPVKSEVSLLKDYL 133 GTGCCCGTGAAGAGCGAGGTGAGCCTGCTGAAGGACTACCTGA RTHLLHVDHRYLILL GGACCCACCTGCTGCACGTGGACCACAGGTACCTGATCCTGCTG DLGFDGTSDRDYEI GACCTGGGCTTCGACGGCACCAGCGACAGGGACTACGAGATCC QTAQLLTGELNFKG AGACCGCCCAGCTGCTGACCGGCGAGCTGAACTTCAAGGGCGCC ARLGDTRKPDVCVY AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCGTGTACTACG YGEDGLIIDNKAYG GCGAGGACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGG KGYSLPIKQADEMY CTACAGCCTGCCCATCAAGCAGGCCGACGAGATGTACAGGTACA RYIEENKERNEKLNP TCGAGGAGAACAAGGAGAGGAACGAGAAGCTGAACCCCAACAA NKWWEIFDNDVIHY GTGGTGGGAGATCTTCGACAACGACGTGATCCACTACCACTTCG HFAFISGAFTGGFKE CCTTCATCAGCGGCGCCTTCACCGGCGGCTTCAAGGAGAGGCTG RLDNIRMRSGIYGA GACAACATCAGGATGAGGAGCGGCATCTACGGCGCCGCCGTGA AVNSMNLLLMAEEL ACAGCATGAACCTGCTGCTGATGGCCGAGGAGCTGAAGAGCGG KSGRLGYKECFKLF CAGGCTGGGCTACAAGGAGTGCTTCAAGCTGTTCGACTGCAACG DCNDEIVL ACGAGATCGTGCTG 53 VPVKSEVSLCKDYL 134 GTGCCCGTGAAGAGCGAGGTGAGCCTGTGCAAGGACTACCTGA RSHLNHVDHKYLIL GGAGCCACCTGAACCACGTGGACCACAAGTACCTGATCCTGCTG LDLGFDGTSDRDYEI GACCTGGGCTTCGACGGCACCAGCGACAGGGACTACGAGATCC QTAQLLTGELNFKG AGACCGCCCAGCTGCTGACCGGCGAGCTGAACTTCAAGGGCGCC ARLGDTRKPDVCVY AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCGTGTACTACG YGEDGLIIDNKAYG GCGAGGACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGG KGYSLPIKQADEMY CTACAGCCTGCCCATCAAGCAGGCCGACGAGATGTACAGGTACA RYIEENKERNEKLNP TCGAGGAGAACAAGGAGAGGAACGAGAAGCTGAACCCCAACAA NKWWEIFDNDVIHY GTGGTGGGAGATCTTCGACAACGACGTGATCCACTACCACTTCG HFAFVSGAFTGGFR CCTTCGTGAGCGGCGCCTTCACCGGCGGCTTCAGGGAGAGGCTG ERLENIRMRSGIYGA GAGAACATCAGGATGAGGAGCGGCATCTACGGCGCCGCCGTGA AVNSMNLLLMAEEL ACAGCATGAACCTGCTGCTGATGGCCGAGGAGCTGAAGAGCGG KSGRLGYKECFKLF CAGGCTGGGCTACAAGGAGTGCTTCAAGCTGTTCGACTGCAACG DCNDEIVL ACGAGATCGTGCTG 54 VPVKSEMSLLKDYL 135 GTGCCCGTGAAGAGCGAGATGAGCCTGCTGAAGGACTACCTGA RTHLLHVDHRYLILL GGACCCACCTGCTGCACGTGGACCACAGGTACCTGATCCTGCTG DLGFDGASDRDYEI GACCTGGGCTTCGACGGCGCCAGCGACAGGGACTACGAGATCC QTAQLLTGELNFKG AGACCGCCCAGCTGCTGACCGGCGAGCTGAACTTCAAGGGCGCC ARLGDTRKPDVCVY AGGCTGGGCGACACCAGGAAGCCCGACGTGTGCGTGTACTACG YGEDGLIIDNKAYG GCGAGGACGGCCTGATCATCGACAACAAGGCCTACGGCAAGGG KGYSLPIKQADEMY CTACAGCCTGCCCATCAAGCAGGCCGACGAGATGTACAGGTACA RYIEENKERNEKLNP TCGAGGAGAACAAGGAGAGGAACGAGAAGCTGAACCCCAACAA NKWWEIFDNDVIHY GTGGTGGGAGATCTTCGACAACGACGTGATCCACTACCACTTCG HFAFVSGAFTGGFK CCTTCGTGAGCGGCGCCTTCACCGGCGGCTTCAAGGAGAGGCTG ERLDNIRMRSGIYGA GACAACATCAGGATGAGGAGCGGCATCTACGGCGCCGCCGTGA AVNSMNLLLMAEEL ACAGCATGAACCTGCTGCTGATGGCCGAGGAGCTGAAGAGCGG KSGRLGYKECFKLF CAGGCTGGGCTACAAGGAGTGCTTCAAGCTGTTCGACTGCAACG DCNDEIVL ACGAGATCGTGCTG 55 ILVDKEREMRKAKF 136 ATCCTGGTGGACAAGGAGAGGGAGATGAGGAAGGCCAAGTTCC LKETVLDSKFISLLD TGAAGGAGACCGTGCTGGACAGCAAGTTCATCAGCCTGCTGGAC LAADATKSRDFEIVT CTGGCCGCCGACGCCACCAAGAGCAGGGACTTCGAGATCGTGA AELFKEAYNLNSVL CCGCCGAGCTGTTCAAGGAGGCCTACAACCTGAACAGCGTGCTG LGGSNKPDGLVFTD CTGGGCGGCAGCAACAAGCCCGACGGCCTGGTGTTCACCGACG DFGILLDTKAYKNG ACTTCGGCATCCTGCTGGACACCAAGGCCTACAAGAACGGCTTC FSIYAKDRDQMIRY AGCATCTACGCCAAGGACAGGGACCAGATGATCAGGTACGTGG VDDNNKRDKIRNPN ACGACAACAACAAGAGGGACAAGATCAGGAACCCCAACGAGTG EWWKSFSPLIPNDKF GTGGAAGAGCTTCAGCCCCCTGATCCCCAACGACAAGTTCTACT YYLWVSNFFKGQFK ACCTGTGGGTGAGCAACTTCTTCAAGGGCCAGTTCAAGAACCAG NQIEYVNRETNTYG ATCGAGTACGTGAACAGGGAGACCAACACCTACGGCGCCGTGC AVLNVEQLLYGADA TGAACGTGGAGCAGCTGCTGTACGGCGCCGACGCCGTGATCAAG VIKGIINPNKLHEYFS GGCATCATCAACCCCAACAAGCTGCACGAGTACTTCAGCAACGA NDEIKF CGAGATCAAGTTC 56 TVDEKERLELKEYFI 137 ACCGTGGACGAGAAGGAGAGGCTGGAGCTGAAGGAGTACTTCA SNTRIPSKYITLLDLA TCAGCAACACCAGGATCCCCAGCAAGTACATCACCCTGCTGGAC YDGNANRDFEIVTA CTGGCCTACGACGGCAACGCCAACAGGGACTTCGAGATCGTGAC ELFKDIFKLQSKHM CGCCGAGCTGTTCAAGGACATCTTCAAGCTGCAGAGCAAGCACA GGTRKPDILIWTDKF TGGGCGGCACCAGGAAGCCCGACATCCTGATCTGGACCGACAA GVIADTKAYSKGYK GTTCGGCGTGATCGCCGACACCAAGGCCTACAGCAAGGGCTACA KNISEADKMVRYVN AGAAGAACATCAGCGAGGCCGACAAGATGGTGAGGTACGTGAA ENTNRNKVDNTNE CGAGAACACCAACAGGAACAAGGTGGACAACACCAACGAGTGG WWNSFDSRIPKDAY TGGAACAGCTTCGACAGCAGGATCCCCAAGGACGCCTACTACTT YFLWISSEFVGKFDE CCTGTGGATCAGCAGCGAGTTCGTGGGCAAGTTCGACGAGCAGC QLTETSSRTGRNGAS TGACCGAGACCAGCAGCAGGACCGGCAGGAACGGCGCCAGCAT INVYQLLRGADLVQ CAACGTGTACCAGCTGCTGAGGGGCGCCGACCTGGTGCAGAAG KSKFNIHDLPNLMQ AGCAAGTTCAACATCCACGACCTGCCCAACCTGATGCAGAACAA NNEIKF CGAGATCAAGTTC 57 TLQKSDIEKFKNQLR 138 ACCCTGCAGAAGAGCGACATCGAGAAGTTCAAGAACCAGCTGA TELTNIDHSYLKGIDI GGACCGAGCTGACCAACATCGACCACAGCTACCTGAAGGGCAT ASKKTTTNVENTEF CGACATCGCCAGCAAGAAGACCACCACCAACGTGGAGAACACC EAISTKVFTDELGFF GAGTTCGAGGCCATCAGCACCAAGGTGTTCACCGACGAGCTGGG GEHLGGSNKPDGLI CTTCTTCGGCGAGCACCTGGGCGGCAGCAACAAGCCCGACGGCC WDNDCAIILDSKAY TGATCTGGGACAACGACTGCGCCATCATCCTGGACAGCAAGGCC SEGFPLTASHTDAM TACAGCGAGGGCTTCCCCCTGACCGCCAGCCACACCGACGCCAT GRYLRQFKERKEEIK GGGCAGGTACCTGAGGCAGTTCAAGGAGAGGAAGGAGGAGATC PTWWDIAPDNLANT AAGCCCACCTGGTGGGACATCGCCCCCGACAACCTGGCCAACAC YFAYVSGSFSGNYK CTACTTCGCCTACGTGAGCGGCAGCTTCAGCGGCAACTACAAGG AQLQKFRQDTNHM CCCAGCTGCAGAAGTTCAGGCAGGACACCAACCACATGGGCGG GGALEFVKLLLLAN CGCCCTGGAGTTCGTGAAGCTGCTGCTGCTGGCCAACAACTACA NYKAHKMSINEVKE AGGCCCACAAGATGAGCATCAACGAGGTGAAGGAGAGCATCCT SILDYNISY GGACTACAACATCAGCTAC 58 VKEKTDAALVKERV 139 GTGAAGGAGAAGACCGACGCCGCCCTGGTGAAGGAGAGGGTGA RLQLHNINHKYLALI GGCTGCAGCTGCACAACATCAACCACAAGTACCTGGCCCTGATC DYAFSGKNNSRDFE GACTACGCCTTCAGCGGCAAGAACAACAGCAGGGACTTCGAGG VYTIDLLVNELTFGG TGTACACCATCGACCTGCTGGTGAACGAGCTGACCTTCGGCGGC LHLGGTRKPDGIFY CTGCACCTGGGCGGCACCAGGAAGCCCGACGGCATCTTCTACCA HGSNGIIIDNKAYAK CGGCAGCAACGGCATCATCATCGACAACAAGGCCTACGCCAAG GFVITRNMADEMIR GGCTTCGTGATCACCAGGAACATGGCCGACGAGATGATCAGGTA YVQENNDRNPERNP CGTGCAGGAGAACAACGACAGGAACCCCGAGAGGAACCCCAAC NCWWKGFPHDVTR TGCTGGTGGAAGGGCTTCCCCCACGACGTGACCAGGTACAACTA YNYVFISSMFKGEV CGTGTTCATCAGCAGCATGTTCAAGGGCGAGGTGGAGCACATGC EHMLDNIRQSTGIDG TGGACAACATCAGGCAGAGCACCGGCATCGACGGCTGCGTGCT CVLTIENLLYYADAI GACCATCGAGAACCTGCTGTACTACGCCGACGCCATCAAGGGCG KGGTLSKATFINGFN GCACCCTGAGCAAGGCCACCTTCATCAACGGCTTCAACGCCAAC ANKEMVF AAGGAGATGGTGTTC 59 VKETTDSVIIKDRVR 140 GTGAAGGAGACCACCGACAGCGTGATCATCAAGGACAGGGTGA LKLHHVNHKYLTLI GGCTGAAGCTGCACCACGTGAACCACAAGTACCTGACCCTGATC DYAFSGKNNCMDFE GACTACGCCTTCAGCGGCAAGAACAACTGCATGGACTTCGAGGT VYTIDLLVNELAFN GTACACCATCGACCTGCTGGTGAACGAGCTGGCCTTCAACGGCG GVHLGGTRKPDGIF TGCACCTGGGCGGCACCAGGAAGCCCGACGGCATCTTCTACCAC YHNRNGIIIDNKAYS AACAGGAACGGCATCATCATCGACAACAAGGCCTACAGCCACG HGFTLSRAMADEMI GCTTCACCCTGAGCAGGGCCATGGCCGACGAGATGATCAGGTAC RYIQENNDRNPERN ATCCAGGAGAACAACGACAGGAACCCCGAGAGGAACCCCAACA PNKWWENFDKGVN AGTGGTGGGAGAACTTCGACAAGGGCGTGAACCAGTTCAACTTC QFNFVFISSLFKGEIE GTGTTCATCAGCAGCCTGTTCAAGGGCGAGATCGAGCACATGCT HMLTNIKQSTDGVE GACCAACATCAAGCAGAGCACCGACGGCGTGGAGGGCTGCGTG GCVLSAENLLYFAE CTGAGCGCCGAGAACCTGCTGTACTTCGCCGAGGCCATGAAGAG AMKSGVMPKTEFIS CGGCGTGATGCCCAAGACCGAGTTCATCAGCTACTTCGGCGCCG YFGAGKEIQF GCAAGGAGATCCAGTTC 60 SACKADITELKDKIR 141 AGCGCCTGCAAGGCCGACATCACCGAGCTGAAGGACAAGATCA KSLKVLDHKYLVLV GGAAGAGCCTGAAGGTGCTGGACCACAAGTACCTGGTGCTGGT DLAYSDASTKSKKN GGACCTGGCCTACAGCGACGCCAGCACCAAGAGCAAGAAGAAC SDAREFEIQTADLFT AGCGACGCCAGGGAGTTCGAGATCCAGACCGCCGACCTGTTCAC KELKFDGMRLGDSN CAAGGAGCTGAAGTTCGACGGCATGAGGCTGGGCGACAGCAAC RPDVIISHDNFGTIID AGGCCCGACGTGATCATCAGCCACGACAACTTCGGCACCATCAT NKSYKDGFNIDKKC CGACAACAAGAGCTACAAGGACGGCTTCAACATCGACAAGAAG ADEMSRYINENQRRI TGCGCCGACGAGATGAGCAGGTACATCAACGAGAACCAGAGGA PELPKNEWWKNFD GGATCCCCGAGCTGCCCAAGAACGAGTGGTGGAAGAACTTCGA VNVDIFTFLFITSYLK CGTGAACGTGGACATCTTCACCTTCCTGTTCATCACCAGCTACCT GNFKDQLEYISKSQS GAAGGGCAACTTCAAGGACCAGCTGGAGTACATCAGCAAGAGC DIKGAAISVEHLLYI CAGAGCGACATCAAGGGCGCCGCCATCAGCGTGGAGCACCTGC SEKVKNGSMDKADF TGTACATCAGCGAGAAGGTGAAGAACGGCAGCATGGACAAGGC FKLFNNDEIRV CGACTTCTTCAAGCTGTTCAACAACGACGAGATCAGGGTG 61 VLKDKHLEKIKEKF 142 GTGCTGAAGGACAAGCACCTGGAGAAGATCAAGGAGAAGTTCC LENTSLDPRFISLIEIS TGGAGAACACCAGCCTGGACCCCAGGTTCATCAGCCTGATCGAG RDKKQNRAFEIITAE ATCAGCAGGGACAAGAAGCAGAACAGGGCCTTCGAGATCATCA LFNTSYNLSAIHLGG CCGCCGAGCTGTTCAACACCAGCTACAACCTGAGCGCCATCCAC GRRPDVLAYNDNFG CTGGGCGGCGGCAGGAGGCCCGACGTGCTGGCCTACAACGACA IIVDTKAYKNGYGR ACTTCGGCATCATCGTGGACACCAAGGCCTACAAGAACGGCTAC NVNQEDEMVRYITE GGCAGGAACGTGAACCAGGAGGACGAGATGGTGAGGTACATCA NKIRKQDISKNNWW CCGAGAACAAGATCAGGAAGCAGGACATCAGCAAGAACAACTG KYFSKSIPSTSYYHL GTGGAAGTACTTCAGCAAGAGCATCCCCAGCACCAGCTACTACC WISSEFVGMFSDQL ACCTGTGGATCAGCAGCGAGTTCGTGGGCATGTTCAGCGACCAG RETSSRTGENGGAM CTGAGGGAGACCAGCAGCAGGACCGGCGAGAACGGCGGCGCCA NVEQLLIGANQVLN TGAACGTGGAGCAGCTGCTGATCGGCGCCAACCAGGTGCTGAAC NVLDPNRLPEYMEN AACGTGCTGGACCCCAACAGGCTGCCCGAGTACATGGAGAACA KEIIF AGGAGATCATCTTC 62 ALKDKHLEKIKEKF 143 GCCCTGAAGGACAAGCACCTGGAGAAGATCAAGGAGAAGTTCC LENTSLDPRFISLIEIS TGGAGAACACCAGCCTGGACCCCAGGTTCATCAGCCTGATCGAG RDKKQNRAFEIITAE ATCAGCAGGGACAAGAAGCAGAACAGGGCCTTCGAGATCATCA LFNTSYKLSATHLG CCGCCGAGCTGTTCAACACCAGCTACAAGCTGAGCGCCACCCAC GGRRPDVLVYNDNF CTGGGCGGCGGCAGGAGGCCCGACGTGCTGGTGTACAACGACA GIIVDTKAYKDGYG ACTTCGGCATCATCGTGGACACCAAGGCCTACAAGGACGGCTAC RNVNQEDEMVRYIT GGCAGGAACGTGAACCAGGAGGACGAGATGGTGAGGTACATCA ENNIRKQDINKNDW CCGAGAACAACATCAGGAAGCAGGACATCAACAAGAACGACTG WKYFSKSIPSTSYYH GTGGAAGTACTTCAGCAAGAGCATCCCCAGCACCAGCTACTACC LWISSQFVGMFSDQ ACCTGTGGATCAGCAGCCAGTTCGTGGGCATGTTCAGCGACCAG LRETSSRTGENGGA CTGAGGGAGACCAGCAGCAGGACCGGCGAGAACGGCGGCGCCA MNVEQLLIGANQVL TGAACGTGGAGCAGCTGCTGATCGGCGCCAACCAGGTGCTGAAC NNVLDPNCLPKYME AACGTGCTGGACCCCAACTGCCTGCCCAAGTACATGGAGAACAA NKEIIF GGAGATCATCTTC 63 VLEKSDIEKFKNQLR 144 GTGCTGGAGAAGAGCGACATCGAGAAGTTCAAGAACCAGCTGA TELTNIDHSYLKGIDI GGACCGAGCTGACCAACATCGACCACAGCTACCTGAAGGGCAT ASKKKTSNVENTEF CGACATCGCCAGCAAGAAGAAGACCAGCAACGTGGAGAACACC EAISTKIFTDELGFSG GAGTTCGAGGCCATCAGCACCAAGATCTTCACCGACGAGCTGGG KHLGGSNKPDGLLW CTTCAGCGGCAAGCACCTGGGCGGCAGCAACAAGCCCGACGGC DDDCAIILDSKAYSE CTGCTGTGGGACGACGACTGCGCCATCATCCTGGACAGCAAGGC GFPLTASHTDAMGR CTACAGCGAGGGCTTCCCCCTGACCGCCAGCCACACCGACGCCA YLRQFTERKEEIKPT TGGGCAGGTACCTGAGGCAGTTCACCGAGAGGAAGGAGGAGAT WWDIAPEHLDNTYF CAAGCCCACCTGGTGGGACATCGCCCCCGAGCACCTGGACAACA AYVSGSFSGNYKEQ CCTACTTCGCCTACGTGAGCGGCAGCTTCAGCGGCAACTACAAG LQKFRQDTNHLGGA GAGCAGCTGCAGAAGTTCAGGCAGGACACCAACCACCTGGGCG LEFVKLLLLANNYK GCGCCCTGGAGTTCGTGAAGCTGCTGCTGCTGGCCAACAACTAC TQKMSKKEVKKSIL AAGACCCAGAAGATGAGCAAGAAGGAGGTGAAGAAGAGCATCC DYNISY TGGACTACAACATCAGCTAC 64 AEADVTSEKIKNHF 145 GCCGAGGCCGACGTGACCAGCGAGAAGATCAAGAACCACTTCA RRVTELPERYLELLD GGAGGGTGACCGAGCTGCCCGAGAGGTACCTGGAGCTGCTGGA IAFDHKRNRDFEMV CATCGCCTTCGACCACAAGAGGAACAGGGACTTCGAGATGGTG TAGLFKDVYGLESV ACCGCCGGCCTGTTCAAGGACGTGTACGGCCTGGAGAGCGTGCA HLGGANKPDGVVY CCTGGGCGGCGCCAACAAGCCCGACGGCGTGGTGTACAACGAC NDNFGIILDTKAYEN AACTTCGGCATCATCCTGGACACCAAGGCCTACGAGAACGGCTA GYGKHISQIDEMVR CGGCAAGCACATCAGCCAGATCGACGAGATGGTGAGGTACATC YIDDNRLRDTTRNP GACGACAACAGGCTGAGGGACACCACCAGGAACCCCAACAAGT NKWWENFDADIPSD GGTGGGAGAACTTCGACGCCGACATCCCCAGCGACCAGTTCTAC QFYYLWVSGKFLPN TACCTGTGGGTGAGCGGCAAGTTCCTGCCCAACTTCGCCGAGCA FAEQLKQTNYRSHA GCTGAAGCAGACCAACTACAGGAGCCACGCCAACGGCGGCGGC NGGGLEVQQLLLGA CTGGAGGTGCAGCAGCTGCTGCTGGGCGCCGACGCCGTGAAGA DAVKRRKLDVNTIP GGAGGAAGCTGGACGTGAACACCATCCCCAACTACATGAAGAA NYMKNEVITL CGAGGTGATCACCCTG 65 AEADLNSEKIKNHY 146 GCCGAGGCCGACCTGAACAGCGAGAAGATCAAGAACCACTACA RKITNLPEKYIELLDI GGAAGATCACCAACCTGCCCGAGAAGTACATCGAGCTGCTGGA AFDHRRHQDFEIVT CATCGCCTTCGACCACAGGAGGCACCAGGACTTCGAGATCGTGA AGLFKDCYGLSSIHL CCGCCGGCCTGTTCAAGGACTGCTACGGCCTGAGCAGCATCCAC GGQNKPDGVVFNN CTGGGCGGCCAGAACAAGCCCGACGGCGTGGTGTTCAACAACA KFGIILDTKAYEKGY AGTTCGGCATCATCCTGGACACCAAGGCCTACGAGAAGGGCTAC GMHIGQIDEMCRYI GGCATGCACATCGGCCAGATCGACGAGATGTGCAGGTACATCG DDNKKRDIVRQPNE ACGACAACAAGAAGAGGGACATCGTGAGGCAGCCCAACGAGTG WWKNFGDNIPKDQF GTGGAAGAACTTCGGCGACAACATCCCCAAGGACCAGTTCTACT YYLWISGKFLPRFNE ACCTGTGGATCAGCGGCAAGTTCCTGCCCAGGTTCAACGAGCAG QLKQTHYRTSINGG CTGAAGCAGACCCACTACAGGACCAGCATCAACGGCGGCGGCC GLEVSQLLLGANAA TGGAGGTGAGCCAGCTGCTGCTGGGCGCCAACGCCGCCATGAA MKGKLDVNTLPKH GGGCAAGCTGGACGTGAACACCCTGCCCAAGCACATGAACAAC MNNQVIKL CAGGTGATCAAGCTG 66 VLKDAALQKTKNTL 147 GTGCTGAAGGACGCCGCCCTGCAGAAGACCAAGAACACCCTGC LNELTEIDPADIEVIE TGAACGAGCTGACCGAGATCGACCCCGCCGACATCGAGGTGATC MSWKKATTRSQNTL GAGATGAGCTGGAAGAAGGCCACCACCAGGAGCCAGAACACCC EATLFEVKVVEIFKK TGGAGGCCACCCTGTTCGAGGTGAAGGTGGTGGAGATCTTCAAG YFELNGEHLGGQNR AAGTACTTCGAGCTGAACGGCGAGCACCTGGGCGGCCAGAACA PDGAVYYNSTYGIIL GGCCCGACGGCGCCGTGTACTACAACAGCACCTACGGCATCATC DTKAYSNGYNIPVD CTGGACACCAAGGCCTACAGCAACGGCTACAACATCCCCGTGGA QQREMVDYITDVID CCAGCAGAGGGAGATGGTGGACTACATCACCGACGTGATCGAC KNQNVTPNRWWEA AAGAACCAGAACGTGACCCCCAACAGGTGGTGGGAGGCCTTCC FPATLLKNNIYYLW CCGCCACCCTGCTGAAGAACAACATCTACTACCTGTGGGTGGCC VAGGFTGKYLDQLT GGCGGCTTCACCGGCAAGTACCTGGACCAGCTGACCAGGACCCA RTHNQTNMDGGAM CAACCAGACCAACATGGACGGCGGCGCCATGACCACCGAGGTG TTEVLLRLANKVSS CTGCTGAGGCTGGCCAACAAGGTGAGCAGCGGCAACCTGAAGA GNLKTTDIPKLMTN CCACCGACATCCCCAAGCTGATGACCAACAAGCTGATCCTGAGC KLILS 67 AEADLDSERIKNHY 148 GCCGAGGCCGACCTGGACAGCGAGAGGATCAAGAACCACTACA RKITNLPEKYIELLDI GGAAGATCACCAACCTGCCCGAGAAGTACATCGAGCTGCTGGA AFDHHRHQDFEIITA CATCGCCTTCGACCACCACAGGCACCAGGACTTCGAGATCATCA GLFKDCYGLSSIHLG CCGCCGGCCTGTTCAAGGACTGCTACGGCCTGAGCAGCATCCAC GQNKPDGVVFNGKF CTGGGCGGCCAGAACAAGCCCGACGGCGTGGTGTTCAACGGCA GIILDTKAYEKGYG AGTTCGGCATCATCCTGGACACCAAGGCCTACGAGAAGGGCTAC MHINQIDEMCRYIED GGCATGCACATCAACCAGATCGACGAGATGTGCAGGTACATCG NKQRDKIRQPNEW AGGACAACAAGCAGAGGGACAAGATCAGGCAGCCCAACGAGTG WNNFGDNIPENKFY GTGGAACAACTTCGGCGACAACATCCCCGAGAACAAGTTCTACT YLWVSGKFLPKFNE ACCTGTGGGTGAGCGGCAAGTTCCTGCCCAAGTTCAACGAGCAG QLKQTHYRTGINGG CTGAAGCAGACCCACTACAGGACCGGCATCAACGGCGGCGGCC GLEVSQLLLGADAV TGGAGGTGAGCCAGCTGCTGCTGGGCGCCGACGCCGTGATGAA MKGALNVNILPTYM GGGCGCCCTGAACGTGAACATCCTGCCCACCTACATGCACAACA HNNVIQ ACGTGATCCAG 68 EISDIALQKEKAYFY 149 GAGATCAGCGACATCGCCCTGCAGAAGGAGAAGGCCTACTTCTA KNTALSKRHISILEIA CAAGAACACCGCCCTGAGCAAGAGGCACATCAGCATCCTGGAG FDGSKNRDLEILSAE ATCGCCTTCGACGGCAGCAAGAACAGGGACCTGGAGATCCTGA VFKDYYQLESIHLG GCGCCGAGGTGTTCAAGGACTACTACCAGCTGGAGAGCATCCAC GGLKPDGIAFNQNF CTGGGCGGCGGCCTGAAGCCCGACGGCATCGCCTTCAACCAGAA GIIVDTKAYKGVYS CTTCGGCATCATCGTGGACACCAAGGCCTACAAGGGCGTGTACA RSRAEADKMFRYIE GCAGGAGCAGGGCCGAGGCCGACAAGATGTTCAGGTACATCGA DNKKRDPKRNQSL GGACAACAAGAAGAGGGACCCCAAGAGGAACCAGAGCCTGTGG WWRSFNEHIPANNF TGGAGGAGCTTCAACGAGCACATCCCCGCCAACAACTTCTACTT YFLWISGKFQRNFD CCTGTGGATCAGCGGCAAGTTCCAGAGGAACTTCGACACCCAGA TQINQLNYETGYRG TCAACCAGCTGAACTACGAGACCGGCTACAGGGGCGGCGCCCT GALSARQFLIGADAI GAGCGCCAGGCAGTTCCTGATCGGCGCCGACGCCATCCAGAAG QKGKIDINDLPSYFN GGCAAGATCGACATCAACGACCTGCCCAGCTACTTCAACAACAG NSVISF CGTGATCAGCTTC 69 TSREKSRLNLKEYFV 150 ACCAGCAGGGAGAAGAGCAGGCTGAACCTGAAGGAGTACTTCG SNTNLPNKFITLLDL TGAGCAACACCAACCTGCCCAACAAGTTCATCACCCTGCTGGAC AYDGKANRDFELIT CTGGCCTACGACGGCAAGGCCAACAGGGACTTCGAGCTGATCAC SELFREIYKLNTRHL CAGCGAGCTGTTCAGGGAGATCTACAAGCTGAACACCAGGCAC GGTRKPDILIWNENF CTGGGCGGCACCAGGAAGCCCGACATCCTGATCTGGAACGAGA GIIADTKAYSKGYK ACTTCGGCATCATCGCCGACACCAAGGCCTACAGCAAGGGCTAC KNISEEDKMVRYIDE AAGAAGAACATCAGCGAGGAGGACAAGATGGTGAGGTACATCG NIKRSKDYNPNEWW ACGAGAACATCAAGAGGAGCAAGGACTACAACCCCAACGAGTG KVFDNEISSNNYFYL GTGGAAGGTGTTCGACAACGAGATCAGCAGCAACAACTACTTCT WISSEFIGKFEEQLQ ACCTGTGGATCAGCAGCGAGTTCATCGGCAAGTTCGAGGAGCAG ETAQRTNVKGASIN CTGCAGGAGACCGCCCAGAGGACCAACGTGAAGGGCGCCAGCA VYQLLMGAHKVQT TCAACGTGTACCAGCTGCTGATGGGCGCCCACAAGGTGCAGACC KELNVNSIPKYMNN AAGGAGCTGAACGTGAACAGCATCCCCAAGTACATGAACAACA TEIKF CCGAGATCAAGTTC 70 NCIKDSIIDIKDRVRT 151 AACTGCATCAAGGACAGCATCATCGACATCAAGGACAGGGTGA KLVHLDHKYLALID GGACCAAGCTGGTGCACCTGGACCACAAGTACCTGGCCCTGATC LAFSDADTRTKKNS GACCTGGCCTTCAGCGACGCCGACACCAGGACCAAGAAGAACA DAREFEIQTADLFTK GCGACGCCAGGGAGTTCGAGATCCAGACCGCCGACCTGTTCACC ELSFNGQRLGDSRK AAGGAGCTGAGCTTCAACGGCCAGAGGCTGGGCGACAGCAGGA PDIIISFDKIGTIIDNK AGCCCGACATCATCATCAGCTTCGACAAGATCGGCACCATCATC SYKDGFNISRPCADE GACAACAAGAGCTACAAGGACGGCTTCAACATCAGCAGGCCCT MIRYINENNLRKKSL GCGCCGACGAGATGATCAGGTACATCAACGAGAACAACCTGAG NANEWWNKFDPTIT GAAGAAGAGCCTGAACGCCAACGAGTGGTGGAACAAGTTCGAC AYSFLFITSYLKGQF CCCACCATCACCGCCTACAGCTTCCTGTTCATCACCAGCTACCTG QEQLEYISNANGGIK AAGGGCCAGTTCCAGGAGCAGCTGGAGTACATCAGCAACGCCA GAAIGIENLLYLSEA ACGGCGGCATCAAGGGCGCCGCCATCGGCATCGAGAACCTGCT LKSGKISHKDFYQNF GTACCTGAGCGAGGCCCTGAAGAGCGGCAAGATCAGCCACAAG NNKEITY GACTTCTACCAGAACTTCAACAACAAGGAGATCACCTAC 71 LPQKDQVQQQQDEL 152 CTGCCCCAGAAGGACCAGGTGCAGCAGCAGCAGGACGAGCTGA RPMLKNVDHRYLQL GGCCCATGCTGAAGAACGTGGACCACAGGTACCTGCAGCTGGTG VELALDSDQNSEYS GAGCTGGCCCTGGACAGCGACCAGAACAGCGAGTACAGCCAGT QFEQLTMELVLKHL TCGAGCAGCTGACCATGGAGCTGGTGCTGAAGCACCTGGACTTC DFDGKPLGGSNKPD GACGGCAAGCCCCTGGGCGGCAGCAACAAGCCCGACGGCATCG GIAWDNDGNFIIFDT CCTGGGACAACGACGGCAACTTCATCATCTTCGACACCAAGGCC KAYNKGYSLAGNT TACAACAAGGGCTACAGCCTGGCCGGCAACACCGACAAGGTGA DKVKRYIDDVRDRD AGAGGTACATCGACGACGTGAGGGACAGGGACACCAGCAGGAC TSRTSTWWQLVPKS CAGCACCTGGTGGCAGCTGGTGCCCAAGAGCATCGACGTGCACA IDVHNLLRFVYVSG ACCTGCTGAGGTTCGTGTACGTGAGCGGCAACTTCACCGGCAAC NFTGNYMKLLDSLR TACATGAAGCTGCTGGACAGCCTGAGGAGCTGGAGCAACGCCC SWSNAQGGLASVEK AGGGCGGCCTGGCCAGCGTGGAGAAGCTGCTGCTGACCAGCGA LLLTSELYLRNMYS GCTGTACCTGAGGAACATGTACAGCCACCAGGAGCTGATCGACA HQELIDSWTDNNVK GCTGGACCGACAACAACGTGAAGCAC H 72 TTDAVVVKDRARV 153 ACCACCGACGCCGTGGTGGTGAAGGACAGGGCCAGGGTGAGGC RLHNINHKYLTLIDY TGCACAACATCAACCACAAGTACCTGACCCTGATCGACTACGCC AFSGKNNCTEFEIYT TTCAGCGGCAAGAACAACTGCACCGAGTTCGAGATCTACACCAT IDLLVNELAFNGIHL CGACCTGCTGGTGAACGAGCTGGCCTTCAACGGCATCCACCTGG GGTRKPDGIFDYNQ GCGGCACCAGGAAGCCCGACGGCATCTTCGACTACAACCAGCA QGIIIDNKAYSKGFTI GGGCATCATCATCGACAACAAGGCCTACAGCAAGGGCTTCACCA TRSMADEMVRYVQ TCACCAGGAGCATGGCCGACGAGATGGTGAGGTACGTGCAGGA ENNDRNPERNKTQ GAACAACGACAGGAACCCCGAGAGGAACAAGACCCAGTGGTGG WWLNFGDNVNHFN CTGAACTTCGGCGACAACGTGAACCACTTCAACTTCGTGTTCAT FVFISSMFKGEVRH CAGCAGCATGTTCAAGGGCGAGGTGAGGCACATGCTGAACAAC MLNNIKQSTGVDGC ATCAAGCAGAGCACCGGCGTGGACGGCTGCGTGCTGACCGCCG VLTAENLLYFADAI AGAACCTGCTGTACTTCGCCGACGCCATCAAGGGCGGCACCGTG KGGTVKRTDFINLF AAGAGGACCGACTTCATCAACCTGTTCGGCAAGAACGACGAGCT GKNDEL G 73 LPKKDNVQRQQDEL 154 CTGCCCAAGAAGGACAACGTGCAGAGGCAGCAGGACGAGCTGA RPLLKHVDHRYLQL GGCCCCTGCTGAAGCACGTGGACCACAGGTACCTGCAGCTGGTG VELALDSSQNSEYS GAGCTGGCCCTGGACAGCAGCCAGAACAGCGAGTACAGCATGC MLESMTMELLLTHL TGGAGAGCATGACCATGGAGCTGCTGCTGACCCACCTGGACTTC DFDGASLGGASKPD GACGGCGCCAGCCTGGGCGGCGCCAGCAAGCCCGACGGCATCG GIAWDKDGNFLIVD CCTGGGACAAGGACGGCAACTTCCTGATCGTGGACACCAAGGCC TKAYDNGYSLAGNT TACGACAACGGCTACAGCCTGGCCGGCAACACCGACAAGGTGG DKVARYIDDVRAKD CCAGGTACATCGACGACGTGAGGGCCAAGGACCCCAACAGGGC PNRASTWWTQVPES CAGCACCTGGTGGACCCAGGTGCCCGAGAGCCTGAACGTGGAC LNVDDNLSFMYVSG GACAACCTGAGCTTCATGTACGTGAGCGGCAGCTTCACCGGCAA SFTGNYQRLLKDLR CTACCAGAGGCTGCTGAAGGACCTGAGGGCCAGGACCAACGCC ARTNARGGLTTVEK AGGGGCGGCCTGACCACCGTGGAGAAGCTGCTGCTGACCAGCG LLLTSEAYLAKSGY AGGCCTACCTGGCCAAGAGCGGCTACGGCCACACCCAGCTGCTG GHTQLLNDWTDDNI AACGACTGGACCGACGACAACATCGACCAC DH 74 QIKDKYLEDLKLEL 155 CAGATCAAGGACAAGTACCTGGAGGACCTGAAGCTGGAGCTGT YKKTNLPNKYYEM ACAAGAAGACCAACCTGCCCAACAAGTACTACGAGATGGTGGA VDIAYDGKRNREFEI CATCGCCTACGACGGCAAGAGGAACAGGGAGTTCGAGATCTAC YTSDLMQEIYGFKT ACCAGCGACCTGATGCAGGAGATCTACGGCTTCAAGACCACCCT TLLGGTRKPDVVSY GCTGGGCGGCACCAGGAAGCCCGACGTGGTGAGCTACAGCGAC SDAHGYIIDTKAYA GCCCACGGCTACATCATCGACACCAAGGCCTACGCCAACGGCTA NGYRKEIKQEDEMV CAGGAAGGAGATCAAGCAGGAGGACGAGATGGTGAGGTACATC RYIEDNQLKDVLRN GAGGACAACCAGCTGAAGGACGTGCTGAGGAACCCCAACAAGT PNKWWECFDDAEH GGTGGGAGTGCTTCGACGACGCCGAGCACAAGAAGGAGTACTA KKEYYFLWISSKFV CTTCCTGTGGATCAGCAGCAAGTTCGTGGGCGAGTTCAGCAGCC GEFSSQLQDTSRRTG AGCTGCAGGACACCAGCAGGAGGACCGGCATCAAGGGCGGCGC IKGGAVNIVQLLLG CGTGAACATCGTGCAGCTGCTGCTGGGCGCCCACCTGGTGTACA AHLVYSGEISKDQF GCGGCGAGATCAGCAAGGACCAGTTCGCCGCCTACATGAACAA AAYMNNTEINF CACCGAGATCAACTTC 75 MNPRNEIVIAKHLSG 156 ATGAACCCCAGGAACGAGATCGTGATCGCCAAGCACCTGAGCG GNRPEIVCYHPEDKP GCGGCAACAGGCCCGAGATCGTGTGCTACCACCCCGAGGACAA DHGLILDSKAYKSG GCCCGACCACGGCCTGATCCTGGACAGCAAGGCCTACAAGAGC FTIPSGERDKMVRYI GGCTTCACCATCCCCAGCGGCGAGAGGGACAAGATGGTGAGGT EEYITKNQLQNPNE ACATCGAGGAGTACATCACCAAGAACCAGCTGCAGAACCCCAA WWKNLKGAEYPGI CGAGTGGTGGAAGAACCTGAAGGGCGCCGAGTACCCCGGCATC VGFGFISNSFLGHYR GTGGGCTTCGGCTTCATCAGCAACAGCTTCCTGGGCCACTACAG KQLDYIMRRTKIKG GAAGCAGCTGGACTACATCATGAGGAGGACCAAGATCAAGGGC SSITTEHLLKTVEDV AGCAGCATCACCACCGAGCACCTGCTGAAGACCGTGGAGGACG LSEKGNVIDFFKYFL TGCTGAGCGAGAAGGGCAACGTGATCGACTTCTTCAAGTACTTC E CTGGAG 76 EIKNQEIEELKQIALN 157 GAGATCAAGAACCAGGAGATCGAGGAGCTGAAGCAGATCGCCC KYTALPSEWVELIEI TGAACAAGTACACCGCCCTGCCCAGCGAGTGGGTGGAGCTGATC SRDKDQSTIFEMKV GAGATCAGCAGGGACAAGGACCAGAGCACCATCTTCGAGATGA AELFKTCYRIKSLHL AGGTGGCCGAGCTGTTCAAGACCTGCTACAGGATCAAGAGCCTG GGASKPDCLLWDDS CACCTGGGCGGCGCCAGCAAGCCCGACTGCCTGCTGTGGGACGA FSVIVDAKAYKDGF CAGCTTCAGCGTGATCGTGGACGCCAAGGCCTACAAGGACGGCT PFQASEKDKMVRYL TCCCCTTCCAGGCCAGCGAGAAGGACAAGATGGTGAGGTACCTG RECERKDKAENATE AGGGAGTGCGAGAGGAAGGACAAGGCCGAGAACGCCACCGAGT WWNNFPPELNSNQL GGTGGAACAACTTCCCCCCCGAGCTGAACAGCAACCAGCTGTTC FFMFASSFFSSTAEK TTCATGTTCGCCAGCAGCTTCTTCAGCAGCACCGCCGAGAAGCA HLESVSIASKFSGCA CCTGGAGAGCGTGAGCATCGCCAGCAAGTTCAGCGGCTGCGCCT WDVDNLLSGANFFL GGGACGTGGACAACCTGCTGAGCGGCGCCAACTTCTTCCTGCAG QNPQATLQYHLIRV AACCCCCAGGCCACCCTGCAGTACCACCTGATCAGGGTGTTCAG FSNKVVD CAACAAGGTGGTGGAC 77 LPHKDNVIKQQDEL 158 CTGCCCCACAAGGACAACGTGATCAAGCAGCAGGACGAGCTGA RPMLKHVNHKYLQ GGCCCATGCTGAAGCACGTGAACCACAAGTACCTGCAGCTGGTG LVELAFESSRNSEYS GAGCTGGCCTTCGAGAGCAGCAGGAACAGCGAGTACAGCCAGT QFETLTMELVLKYL TCGAGACCCTGACCATGGAGCTGGTGCTGAAGTACCTGGACTTC DFSGKSLGGANKPD AGCGGCAAGAGCCTGGGCGGCGCCAACAAGCCCGACGGCATCG GIAWDPLGNFLIFDT CCTGGGACCCCCTGGGCAACTTCCTGATCTTCGACACCAAGGCC KAYKHGYTLSNNTD TACAAGCACGGCTACACCCTGAGCAACAACACCGACAGGGTGG RVARYINDVRDKDI CCAGGTACATCAACGACGTGAGGGACAAGGACATCCAGAGGAT QRISRWWQSIPTYID CAGCAGGTGGTGGCAGAGCATCCCCACCTACATCGACGTGAAG VKNKLQFVYISGSFT AACAAGCTGCAGTTCGTGTACATCAGCGGCAGCTTCACCGGCCA GHYLRLLNDLRSRT CTACCTGAGGCTGCTGAACGACCTGAGGAGCAGGACCAGGGCC RAKGGLVTVEKLLL AAGGGCGGCCTGGTGACCGTGGAGAAGCTGCTGCTGACCACCG TTERYLAEADYTHK AGAGGTACCTGGCCGAGGCCGACTACACCCACAAGGAGCTGTTC ELFDDWMDDNIEH GACGACTGGATGGACGACAACATCGAGCAC 78 RISPSNLEQTKQQLR 159 AGGATCAGCCCCAGCAACCTGGAGCAGACCAAGCAGCAGCTGA EELINLDHQYLDILD GGGAGGAGCTGATCAACCTGGACCACCAGTACCTGGACATCCTG FSIAGNVGARQFEV GACTTCAGCATCGCCGGCAACGTGGGCGCCAGGCAGTTCGAGGT RIVELLNEIIIAKHLS GAGGATCGTGGAGCTGCTGAACGAGATCATCATCGCCAAGCACC GGNRPEIIGFNPKEN TGAGCGGCGGCAACAGGCCCGAGATCATCGGCTTCAACCCCAA PEDCIIMDSKAYKEG GGAGAACCCCGAGGACTGCATCATCATGGACAGCAAGGCCTAC FNIPANERDKMIRYV AAGGAGGGCTTCAACATCCCCGCCAACGAGAGGGACAAGATGA EEYNAKDNTLNNNK TCAGGTACGTGGAGGAGTACAACGCCAAGGACAACACCCTGAA WWKNFESPNYPTNQ CAACAACAAGTGGTGGAAGAACTTCGAGAGCCCCAACTACCCC VKFSFVSSSFIGQFT ACCAACCAGGTGAAGTTCAGCTTCGTGAGCAGCAGCTTCATCGG NQLTYINNRTNVNG CCAGTTCACCAACCAGCTGACCTACATCAACAACAGGACCAACG SAITAETLLRKVENV TGAACGGCAGCGCCATCACCGCCGAGACCCTGCTGAGGAAGGT MNVNTEYNLNNFFE GGAGAACGTGATGAACGTGAACACCGAGTACAACCTGAACAAC ELGSNTLVA TTCTTCGAGGAGCTGGGCAGCAACACCCTGGTGGCC 79 TFDSTVADNLKNLIL 160 ACCTTCGACAGCACCGTGGCCGACAACCTGAAGAACCTGATCCT PKLKELDHKYLQAI GCCCAAGCTGAAGGAGCTGGACCACAAGTACCTGCAGGCCATC DIAYKRSNTTNHEN GACATCGCCTACAAGAGGAGCAACACCACCAACCACGAGAACA TLLEVLSADLFTKE CCCTGCTGGAGGTGCTGAGCGCCGACCTGTTCACCAAGGAGATG MDYHGKHLGGANK GACTACCACGGCAAGCACCTGGGCGGCGCCAACAAGCCCGACG PDGFVYDEETGWIL GCTTCGTGTACGACGAGGAGACCGGCTGGATCCTGGACAGCAA DSKAYRDGFAVTAH GGCCTACAGGGACGGCTTCGCCGTGACCGCCCACACCACCGACG TTDAMGRYIDQYRD CCATGGGCAGGTACATCGACCAGTACAGGGACAGGGACGACAA RDDKSTWWEDFPK GAGCACCTGGTGGGAGGACTTCCCCAAGGACCTGCCCCAGACCT DLPQTYFAYVSGFYI ACTTCGCCTACGTGAGCGGCTTCTACATCGGCAAGTACCAGGAG GKYQEQLQDFENRK CAGCTGCAGGACTTCGAGAACAGGAAGCACATGAAGGGCGGCC HMKGGLIEVAKLILL TGATCGAGGTGGCCAAGCTGATCCTGCTGGCCGAGAAGTACAAG AEKYKENKITHDQIT GAGAACAAGATCACCCACGACCAGATCACCCTGCAGATCCTGA LQILNDHISQ ACGACCACATCAGCCAG 80 PLDVVEQMKAELRP 161 CCCCTGGACGTGGTGGAGCAGATGAAGGCCGAGCTGAGGCCCC LLNHVNHRLLAIIDF TGCTGAACCACGTGAACCACAGGCTGCTGGCCATCATCGACTTC SYNMSRGDDKRLED AGCTACAACATGAGCAGGGGCGACGACAAGAGGCTGGAGGACT YTAQIYKLISHDTHL ACACCGCCCAGATCTACAAGCTGATCAGCCACGACACCCACCTG LAGPSRPDVVSVIND CTGGCCGGCCCCAGCAGGCCCGACGTGGTGAGCGTGATCAACG LGIIIDSKAYKQGFNI ACCTGGGCATCATCATCGACAGCAAGGCCTACAAGCAGGGCTTC PQAEEDKMVRYLDE AACATCCCCCAGGCCGAGGAGGACAAGATGGTGAGGTACCTGG SIRRDPAINPTKWWE ACGAGAGCATCAGGAGGGACCCCGCCATCAACCCCACCAAGTG YLGASTEYVFQFVSS GTGGGAGTACCTGGGCGCCAGCACCGAGTACGTGTTCCAGTTCG SFSSGASAKLRQIHR TGAGCAGCAGCTTCAGCAGCGGCGCCAGCGCCAAGCTGAGGCA RSSIEGSIITAKNLLL GATCCACAGGAGGAGCAGCATCGAGGGCAGCATCATCACCGCC LAENFLCTNTINIDL AAGAACCTGCTGCTGCTGGCCGAGAACTTCCTGTGCACCAACAC FRQNNEI CATCAACATCGACCTGTTCAGGCAGAACAACGAGATC 81 QLVPSYITQTKLRLS 162 CAGCTGGTGCCCAGCTACATCACCCAGACCAAGCTGAGGCTGAG GLINYIDHSYFDLID CGGCCTGATCAACTACATCGACCACAGCTACTTCGACCTGATCG LGFDGRQNRLYELRI ACCTGGGCTTCGACGGCAGGCAGAACAGGCTGTACGAGCTGAG VELLNLINSLKALHL GATCGTGGAGCTGCTGAACCTGATCAACAGCCTGAAGGCCCTGC SGGNRPEIIAYSPDV ACCTGAGCGGCGGCAACAGGCCCGAGATCATCGCCTACAGCCCC NPINGVIMDSKSYRG GACGTGAACCCCATCAACGGCGTGATCATGGACAGCAAGAGCT GFNIPNSERDKMIRY ACAGGGGCGGCTTCAACATCCCCAACAGCGAGAGGGACAAGAT INEYNQKNPTLNSN GATCAGGTACATCAACGAGTACAACCAGAAGAACCCCACCCTG RWWENFRAPDYPQS AACAGCAACAGGTGGTGGGAGAACTTCAGGGCCCCCGACTACC PLKYSFVSGNFIGQF CCCAGAGCCCCCTGAAGTACAGCTTCGTGAGCGGCAACTTCATC LNQIQYILTQTGING GGCCAGTTCCTGAACCAGATCCAGTACATCCTGACCCAGACCGG GAITSEKLIEKVNAV CATCAACGGCGGCGCCATCACCAGCGAGAAGCTGATCGAGAAG LNPNISYTINNFFND GTGAACGCCGTGCTGAACCCCAACATCAGCTACACCATCAACAA LGCNRLVQ CTTCTTCAACGACCTGGGCTGCAACAGGCTGGTGCAG

In some embodiments, an endonuclease of the present disclosure can have a sequence of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀X₄₁X₄₂X₄₃KX₄₄X₄₅X₄₆X₄₇X₄₈X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅GX₅₆HLGGX₅₇RX₅₈PDGX₅₉X₆₀X₆₁X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂X₇₃X₇₄GX₇₅₁X₇₆DTKX₇₇YX₇₈X₇₉GYX₈₀L PIX₈₁QX₈₂DEMX₈₃RYX₈₄X₈₅ENX₈₆X₈₇RX₈₈X₈₉X₉₀X₉₁NX₉₂NX₉₃WWX₉₄X₉₅X₉₆X₉₇X₉₈X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆FX₁₀₇X₁₀₈X₁₀₉X₁₁₀FX₁₁₁GX₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈RX₁₁₉X₁₂₀X₁₂₁X₁₂₂X₁₂₃X₁₂₄X₁₂₅X₁₂₆GX₁₂₇X₁₂₈X₁₂₉X₁₃₀X₁₃₁X₁₃₂X₁₃₃LLX₁₃₄X₁₃₅X₁₃₆X₁₃₇X₁₃₈X₁₃₉X₁₄₀X₁₄₁X₁₄₂X₁₄₃X₁₄₄X₁₄₅X₁₄₆X₁₄₇X₁₄₈X₁₄₉X₁₅₀X₁₅₁X₁₅₂X₁₅₃FX₁₅₄X₁₅₅X₁₅₆X₁₅₇X₁₅₈X₁₅₉X₁₆₀ (SEQ ID NO: 316), wherein X₁ is F, Q, N, D, or absent, X₂ is L, I, T, S, N, or absent, X₃ is V, I, G, A, E, T, or absent, X₄ is K, C, or absent, X₅ is G, S, or absent, X₆ is A, S, E, D, N, or absent, X₇ is M, I, V, Q, F, L, or absent, X₈ is E, S, T, N, or absent, X₉ is I, M, E, T, Q, or absent, X₁₀ is K, S, L, I, T, E, or absent, X₁₁ is K or absent, X₁₂ is S, A, E, D, or absent, X₁₃ is E, N, Q, K, or absent, X₁₄ is L, M, V, or absent, X₁₅ is R or absent, X₁₆ is H, D, T, G, E, N, or absent, X₁₇ is K, N, Q, E, A, or absent, X₁₈ is L or absent, X₁₉ is R, Q, N, T, D, or absent, X₂₀ is H, M, V, N, T, or absent, X₂₁ is V, L, I, or absent, X₂₂ is P, S, or absent, X₂₃ is H or absent, X₂₄ is E, D, or absent, X₂₅ is Y or absent, X₂₆ is I, L, or absent, X₂₇ is E, Q, G, S, A, Y, or absent, X₂₈ is L or absent, X₂₉ is I, V, L, or absent, X₃₀ is E, D, or absent, X₃₁ is I, L, or absent, X₃₂ is A, S, or absent, X₃₃ is Q, Y, F, or absent, X₃₄ is D or absent, X₃₅ is S, P, or absent, X₃₆ is K, Y, Q, T, or absent, X₃₇ is Q or absent, X₃₈ is N or absent, X₃₉ is R, K, or absent, X₄₀ is L, I, or absent, X₄₁ is L, F, or absent, X₄₂ is E or absent, X₄₃ is F, M, L, or absent, X₄₄ is V, T, or I, X₄₅ 1 S V, M, L, or I, X₄₆ is E, D, or Q, X₄₇ is F or L, X₄₈ is F or L, X₄₀ is K, I, T, or V, X₅₀ is K, N, or E, X₅₁ is I or E, X₅₂ is Y, F, or C, X₅₃ is G, or N, X₅₄ is Y, or F, X₅₅ is R, S, N, E, K, or Q, X₅₆ is K, S, L, V, or T, X₅₇ is S, A, or V, X₅₈ is K or R, X₅₉ is A, I, or V, X₆₀ is L, M, V, I, or C, X₆₁ is F or Y, X₆₂ is T, A, or S, X₆₃ is K, E, or absent, X₆₄ is D, E, or absent, X₆₅ is E, A, or absent, X₆₆ is N, K, or absent, X₆₇ is E, S, or absent, X₆₈ is D, E, Q, A, or absent, X₆₉ is G, V, K, N, or absent, X₇₀ is L, G, E, S, or absent, X₇₁ is V, S, K, T, E, or absent, X₇₂ is L, H, K, E, Y, D, or A, X₇₃ is N, G, or D, X₇₄ is H, F, or Y, X₇₅ is I, or V, X₇₆ is L, V, or I, X₇₇ is A or S, X₇₈ is K or S, X₇₉ is D, G, K, S, or N, X₈₀ is R, N, S, or G, X₈₁ is S, A, or G, X₈₂ is A, I, or V, X₈₃ is Q, E, I, or V, X₈₄ is V or I, X₈₅ is D, R, G, I, or E, X₈₆ is N, I, or Q, X₈₇ is K, D, T, E, or K, X₈₈ is S, N, D, or E, X₈₉ is Q, E, I, K, or A, X₉₀ is V, H, R, K, L, or E, X₉₁ is I, V, or R, X₉₂ is P, S, T, or R, X₉₃ is E, R, C, Q, or K, X₉₄ is E, N, or K, X₉₅ is I, V, N, E, or A, X₉₆ is Y or F, X₉₇ is P, G, or E, X₉₈ is T, E, S, D, K, or N, X₉₉ is S, D, K, G, N, or T, X₁₀₀ is I, T, V, or L, X₁₀₁ is T, N, G, or D, X₁₀₂ is D, E, T, K, or I, X₁₀₃ is F or Y, X₁₀₄ is K or Y, X₁₀₅ is F or Y, X₁₀₆ is L, S, or M, X₁₀₇ is V or I, X₁₀₈ is S or A, X₁₀₉ is G or A, X₁₁₀ is F, Y, H, E, or K, Xiii is Q, K, T, N, or I, X₁₁₂ is D, N, or K, X₁₁₃ is Y, F, I, or V, X₁₁₄ is R, E, K, Q, or F, X₁₁₅ is K, E, A, or N, X₁₁₆ is Q or K, X₁₁₇ is L or I, X₁₁₈ is E, D, N, or Q, X₁₁₉ is V, I, or L, X₁₂₀ is S, N, F, T, or Q, X₁₂₁ is H, I, C, or R, X₁₂₂ is L, D, N, S, or F, X₁₂₃ is T or K, X₁₂₄ is K, G, or N, X₁₂₅ is C, V, or I, X₁₂₆ is Q, L, K, or Y, X₁₂₇ is A, G, or N, X₁₂₈ is V or A, X₁₂₉ is M, L, I, V, or A, X₁₃₀ is S, T, or D, X₁₃₁ is V or I, X₁₃₂ is E, Q, K, S, or I, X₁₃₃ is Q, H, or T, X₁₃₄ is L, R, or Y, X₁₃₅ is G, I, L, or T, X₁₃₆ is G, A, or V, X₁₃₇ is E, N, or D, X₁₃₈ is K, Y, D, E, A, or R, X₁₃₉ is I, F, Y, or C, X₁₄₀ is K or R, X₁₄₁ is E, R, A, G, or T, X₁₄₂ is G or N, X₁₄₃ is S, K, R, or E, X₁₄₄ is L, I, or M, X₁₄₅ is T, S, D, or K, X₁₄₆ is L, H, Y, R, T, or F, X₁₄₇ is E, Y, M, A, or L, X₁₄₈ is E, D, R, or G, X₁₄₉ is V, F, M, L, or I, X₁₅₀ is G, K, R, L, V, or E, X₁₅₁ is K, N, D, L, H, or S, X₁₅₂ is K, L, C, or absent, X₁₅₃ is K, S, I, Y, M, or F, X₁₅₄ is K, L, C, H, D, Q, or N, X₁₅₅ is N or Y, X₁₅₆ is D, K, T, E, C, or absent, X₁₅₇ is E, V, R, or absent, X₁₅₈ is I, F, L, or absent, X₁₅₉ is V, Q, E, L, or absent, and X₁₆₀ is F or absent.

In some embodiments, an endonuclease of the present disclosure can have a sequence of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀X₄₁X₄₂X₄₃KX₄₄X₄₅X₄₆X₄₇X₄₈X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅GX₅₆HLGGX₅₇RX₅₈PDGX₅₉X₆₀X₆₁X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂X₇₃X₇₄GX₇₅IX₇₆DTKX₇₇YX₇₈X₇₉GYX₈₀L PIX₈₁QX₈₂DEMX₈₃RYX₈₄X₈₅ENX₈₆X₈₇RX₈₈X₈₉X₉₀X₉₁NX₉₂NX₉₃WWX₉₄X₉₅X₉₆X₉₇X₉₈X₉₉X₁₀₀X₁₀₁X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆FX₁₀₇X₁₀₈X₁₀₉X₁₁₀FX₁₁₁GX₁₁₂X₁₁₃X₁₁₄X₁₁₅X₁₁₆X₁₁₇X₁₁₈RX₁₁₉X₁₂₀X₁₂₁X₁₂₂X₁₂₃X₁₂₄X₁₂₅X₁₂₆GX₁₂₇X₁₂₈X₁₂₉X₁₃₀X₁₃₁X₁₃₂X₁₃₃LLX₁₃₄X₁₃₅X₁₃₆X₁₃₇X₁₃₈X₁₃₉X₁₄₀X₁₄₁X₁₄₂X₁₄₃X₁₄₄X₁₄₅X₁₄₆X₁₄₇X₁₄₈X₁₄₉X₁₅₀X₁₅₁X₁₅₂X₁₅₃FX₁₅₄X₁₅₅X₁₅₆X₁₅₇X₁₅₈X₁₅₉X₁₆₀ (SEQ ID NO: 317), wherein X₁ is F, Q, N, or absent, X₂ is L, I, T, S, or absent, X₃ is V, I, G, A, E, T, or absent, X₄ is K, C, or absent, X₅ is G, S, or absent, X₆ is A, S, E, D, or absent, X₇ is M, I, V, Q, F, L, or absent, X₈ is E, S, T, or absent, X₉ is I, M, E, T, Q, or absent, X₁₀ is K, S, L, I, T, E, or absent, X₁₁ is K or absent, X₁₂ is S, A, E, D, or absent, X₁₃ is E, N, Q, K, or absent, X₁₄ is L, M, V, or absent, X₁₅ is R or absent, X₁₆ is H, D, T, G, E, N, or absent, X₁₇ is K, N, Q, E, A, or absent, X₁₈ is L or absent, X₁₉ is R, Q, N, T, D, or absent, X₂₀ is H, M, V, N, T, or absent, X₂₁ is V, L, I, or absent, X₂₂ is P, S, or absent, X₂₃ is H or absent, X₂₄ is E, D, or absent, X₂₅ is Y or absent, X₂₆ is I, L, or absent, X₂₇ is E, Q, G, S, A, or absent, X₂₈ is L or absent, X₂₉ is I, V, L, or absent, X₃₀ is E, D, or absent, X₃₁ is I, L, or absent, X₃₂ is A, S, or absent, X₃₃ is Q, Y, F, or absent, X₃₄ is D or absent, X₃₅ is S, P, or absent, X₃₆ is K, Y, Q, T, or absent, X₃₇ is Q or absent, X₃₈ is N or absent, X₃₉ is R or absent, X₄₀ is L, I, or absent, X₄₁ is L, F, or absent, X₄₂ is E or absent, X₄₃ is F, M, L, or absent, X₄₄ is V, T, or I, X₄₅ is V, M, L, or I, X₄₆ is E, D, or Q, X₄₇ is F or L, X₄₈ is F or L, X₄₉ is K, I, T, or V, X₅₀ is K, N, or E, X₅₁ is I or E, X₅₂ is Y, F, or C, X₅₃ is G, or N, X₅₄ is Y, or F, X₅₅ is R, S, N, E, K, or Q, X₅₆ is K, S, L, V, or T, X₅₇ is S or A, X₅₈ is K or R, X₅₉ is A, I, or V, X₆₀ is L, M, V, I, or C, X₆₁ is F or Y, X₆₂ is T, A, or S, X₆₃ is K, E, or absent, X₆₄ is D, E, or absent, X₆₅ is E, A, or absent, X₆₆ is N, K, or absent, X₆₇ is E, S, or absent, X₆₈ is D, E, Q, A, or absent, X₆₉ is G, V, K, N, or absent, X₇₀ is L, G, E, S, or absent, X₇₁ is V, S, K, T, E, or absent, X₇₂ is L, H, K, E, Y, D, or A, X₇₃ is N, G, or D, X₇₄ is H, F, or Y, X₇₅ is I, or V, X₇₆ is L, V, or I, X₇₇ is A or S, X₇₈ is K or S, X₇₉ is D, G, K, S, or N, X₈₀ is R, N, S, or G, X₈₁ is S, A, or G, X₈₂ is A, I, or V, X₈₃ is Q, E, I, or V, X₈₄ is V or I, X₈₅ is D, R, G, I, or E, X₈₆ is N, I, or Q, X₈₇ is K, D, T, E, or K, X₈₈ is S, N, D, or E, X₈₉ is Q, E, I, K, or A, X₉₀ is V, H, R, K, L, or E, X₉₁ is I, V, or R, X₉₂ is P, S, T, or R, X₉₃ is E, R, C, Q, or K, X₉₄ is E, N, or K, X₉₅ is I, V, N, E, or A, X₉₆ is Y or F, X₉₇ is P, G, or E, X₉₈ is T, E, S, D, K, or N, X₉₉ is S, D, K, G, N, or T, X₁₀₀ is I, T, V, or L, X₁₀₁ is T, N, G, or D, X₁₀₂ is D, E, T, K, or I, X₁₀₃ is F or Y, X₁₀₄ is K or Y, X₁₀₅ is F or Y, X₁₀₆ is L, S, or M, X₁₀₇ is V or I, X₁₀₈ is S or A, X₁₀₉ is G or A, X₁₁₀ is F, Y, H, E, or K, Xiii is Q, K, T, N, or I, X₁₁₂ is D, N, or K, X₁₁₃ is Y, F, I, or V, X₁₁₄ is R, E, K, Q, or F, X₁₁₅ is K, E, A, or N, X₁₁₆ is Q or K, X₁₁₇ is L or I, X₁₁₈ is E, D, N, or Q, X₁₁₉ is V, I, or L, X₁₂₀ is S, N, F, T, or Q, X₁₂₁ is H, I, C, or R, X₁₂₂ is L, D, N, S, or F, X₁₂₃ is T or K, X₁₂₄ is K, G, or N, X₁₂₅ is C, V, or I, X₁₂₆ is Q, L, K, or Y, X₁₂₇ is A, G, or N, X₁₂₈ is V or A, X₁₂₉ is M, L, I, V, or A, X₁₃₀ is S, T, or D, X₁₃₁ is V or I, X₁₃₂ is E, Q, K, S, or I, X₁₃₃ is Q, H, or T, X₁₃₄ is L, R, or Y, X₁₃₅ is G, I, L, or T, X₁₃₆ is G, A, or V, X₁₃₇ is E, N, or D, X₁₃₈ is K, Y, D, E, A, or R, X₁₃₉ is I, F, Y, or C, X₁₄₀ is K or R, X₁₄₁ is E, R, A, G, or T, X₁₄₂ is G or N, X₁₄₃ is S, I, K, R, or E, X₁₄₄ is L, I, or M, X₁₄₅ is T, S, D, or K, X₁₄₆ is L, H, Y, R, or T, X₁₄₇ is E, Y, I, M, or A, X₁₄₈ is E, D, R, or G, X₁₄₉ is V, F, M, L, or I, X₁₅₀ is G, K, R, L, V, or E, X₁₅₁ is K, N, D, L, H, or S, X₁₅₂ is K, L, C, or absent, X₁₅₃ is K, S, I, Y, M, or F, X₁₅₄ is K, L, C, H, D, Q, or N, X₁₅₅ is N or Y, X₁₅₆ is D, K, T, E, C, or absent, X₁₅₇ is E, V, R, or absent, X₁₅₈ is I, F, L, or absent, X₁₅₉ is V, Q, E, L, or absent, and X₁₆₀ is F or absent.

In some embodiments, an endonuclease of the present disclosure can have a sequence of X₁LVKSSX₂EEX₃KEELREKLX₄HLSHEYLX₅LX₆DLAYDSKQNRLFEMKVX₇ELLINECGYX₈G LHLGGSRKPDGIX₉YTEGLKX₁₀NYGIIIDTKAYSDGYNLPISQADEMERYIRENNTRNX₁₁X₁₂V NPNEWWENFPX₁₃NINEFYFLFVSGHFKGNX₁₄EEQLERISIX₁₅TX₁₆IKGAAMSVX₁₇TLLLLAN EIKAGRLX₁₈LEEVX₁₉KYFDNKEIX₂₀F (SEQ ID NO: 318), wherein X₁ is F, Q, N, D, or absent, X₂ is M, I, V, Q, F, L, or absent, X₃ is K, S, L, I, T, E, or absent, X₄ is R, Q, N, T, D, or absent, X₅ is E, Q, G, S, A, Y, or absent, X₆ is I, V, L, or absent, X₇ is V, M, L, or I, X₈ is R, S, N, E, K, or Q, X₉ is L, M, V, I, or C, X₁₀ is L, H, K, E, Y, D, or A, X₁₁ is Q, E, I, K, or A, X₁₂ is V, H, R, K, L, or E, X₁₃ is T, E, S, D, K, or N, X₁₄ is Y, F, I, or V, X₁₅ is L, D, N, S, or F, X₁₆ is K, G, or N, X₁₇ is E, Q, K, S, or I, X₁₈ is T, S, D, or K, X₁₉ is G, K, R, L, V, or E, and X₂₀ is V, Q, E, L, or absent.

In some embodiments, an endonuclease of the present disclosure can have a sequence of X₁LVKSSX₂EEX₃KEELREKLX₄HLSHEYLX₅LX₆DLAYDSKQNRLFEMKVX₇ELLINECGYX₈G LHLGGSRKPDGIX₉YTEGLKX₁₀NYGIIIDTKAYSDGYNLPISQADEMERYIRENNTRNX₁₁X₁₂V NPNEWWENFPX₁₃NINEFYFLFVSGHFKGNX₁₄EEQLERISIX₁₅TX₁₆IKGAAMSVX₁₇TLLLLAN EIKAGRLX₁₈LEEVX₁₉KYFDNKEIX₂₀F (SEQ ID NO: 319), wherein X₁ is F, Q, N, or absent, X₂ is M, I, V, Q, F, L, or absent, X₃ is K, S, L, I, T, E, or absent, X₄ is R, Q, N, T, D, or absent, X₅ is E, Q, G, S, A, or absent, X₆ is I, V, L, or absent, X₇ is V, M, L, or I, X₈ is R, S, N, E, K, or Q, X₉ is L, M, V, I, or C, X₁₀ is L, H, K, E, Y, D, or A, X₁₁ is Q, E, I, K, or A, X₁₂ is V, H, R, K, L, or E, X₁₃ is T, E, S, D, K, or N, X₁₄ is Y, F, I, or V, X₁₅ is L, D, N, S, or F, X₁₆ is K, G, or N, X₁₇ is E, Q, K, S, or I, X₁₈ is T, S, D, or K, X₁₉ is G, K, R, L, V, or E, and X₂₀ is V, Q, E, L, or absent. In some embodiments, a cleavage domain disclosed herein comprises a sequence selected from SEQ ID NO: 316-SEQ ID NO: 319.

In some embodiments, an endonuclease of the present disclosure can have conserved amino acid residues at position 76 (D or E), position 98 (D), and position 100 (K), which together preserve catalytic function. In some embodiments, an endonuclease of the present disclosure can have conserved amino acid residues at position 114 (D) and position 118 (R), which together preserve dimerization of two cleavage domains.

In some embodiments, endonucleases disclosed herein (e.g., SEQ ID NO: 1-SEQ ID NO: 81 (nucleic acid sequences of SEQ ID NO: 82-SEQ ID NO: 162)) can have at least 33.3% divergence from SEQ ID NO: 163 (FokI) and, is immunologically orthogonal to SEQ ID NO: 163 (FokI). In some embodiments, an immunologically orthogonal endonuclease (e.g., SEQ ID NO: 1-SEQ ID NO: 81 (nucleic acid sequences of SEQ ID NO: 82-SEQ ID NO: 162)) can be administered to a patient that has already received, and is thus can have an adverse immune reaction to, FokI. In some embodiments, endonucleases disclosed herein (e.g., SEQ ID NO: 1-SEQ ID NO: 81 (nucleic acid sequences of SEQ ID NO: 82-SEQ ID NO: 162)) can have at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% divergence from SEQ ID NO: 163 (FokI).

In some embodiments, an endonuclease disclosed herein (e.g., SEQ ID NO: 1-SEQ ID NO: 81 (nucleic acid sequences of SEQ ID NO: 82-SEQ ID NO: 162)) can be fused to any nucleic acid binding domain disclosed herein to form a non-naturally occurring fusion protein. This fusion protein can have one or more of the following characteristics: (a) induces greater than 1% indels (insertions/deletions) at a target site; (b) the cleavage domain comprises a molecular weight of less than 23 kDa; (c) the cleavage domain comprises less than 196 amino acids; and (d) capable of cleaving across a spacer region greater than 24 base pairs. In some embodiments, the non-naturally occurring fusion protein can induce greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% indels at the target site. In some embodiments, indels are generated via the non-homologous end joining (NHEJ) pathway upon administration of a genome editing complex disclosed herein to a subject. Indels can be measured using deep sequencing.

In still various embodiments, the functional domain can be a cleavage domain or a repression domain. In some aspects, the cleavage domain comprises at least 33.3% divergence from SEQ ID NO: 163 and is immunologically orthogonal to SEQ ID NO: 163. In further aspects, the polypeptide can comprise one or more of the following characteristics: (a) induces greater than 1% indels at a target site; (b) the cleavage domain comprises a molecular weight of less than 23 kDa; (c) the cleavage domain comprises less than 196 amino acids; (d) capable of cleaving across a spacer region greater than 24 base pairs.

DNA Binding Domains Fused to SEQ ID NO: 1-SEQ ID NO: 81 (Nucleic Acid Sequences of SEQ ID NO: 82-SEQ ID NO: 162)

The present disclosure provides for novel compositions of endonucleases with modular nucleic acid binding domains (e.g., TALEs, RNBDs, or MAP-NBDs) described herein. In some instances the novel endonucleases can be fused to a DNA binding domain from Xanthomonas spp. (TALE), Ralstonia (RNBD), or Legionella (MAP-NBD) resulting in genome editing complexes. A TALEN, RNBD-nuclease, or MAP-NBD-nuclease can include multiple components including the DNA binding domain, an optional linker, and a repressor domain. The genome editing complexes described herein can be used to selectively bind and cleave to a target gene sequence for genome editing purposes. For example, a DNA binding domain from Xanthomonas, Ralstonia, or Legionella of the present disclosure can be used to direct the binding of a genome editing complex to a desired genomic sequence.

The genome editing complexes described herein, comprising a DNA binding domain fused to an endonuclease, can be used to edit genomic loci of interest by binding to a target nucleic acid sequence via the DNA binding domain and cleaving phosphodiester bonds of target double stranded DNA via the endonuclease.

In some aspects, DNA binding domains fused to nucleases can create a site-specific double-stranded DNA break when fused to a nuclease. Such breaks can then be subsequently repaired by cellular machinery, through either homology-dependent repair or non-homologous end joining (NHEJ). Genome editing, using DNA binding domains fused to nucleases described herein, can thus be used to delete a sequence of interest (e.g., an aberrantly expressed or mutated gene) or to introduce a nucleic acid sequence of interest (e.g., a functional gene). DNA binding domains of the present disclosure can be programmed to delivery virtually any nuclease, including those disclosed herein, to any target site for therapeutic purposes, including ex vivo engineered cell therapies obtained using the compositions disclosed herein or gene therapy by direct in vivo administration of the compositions disclosed herein. In addition, the DNA binding domain can bind to specific DNA sequences and in some cases they can activate the expression of host genes. In some instances, the disclosure provides for enzymes, e.g., SEQ ID NO: 1-SEQ ID NO: 81 (or any one of nucleic acid sequences of SEQ ID NO: 82-SEQ ID NO: 162) that can be fused to the DNA binding domains of TALEs, RNBDs, and MAP-NBDs. In some instances, enzymes of the disclosure, including SEQ ID NO: 1 (nucleic acid sequence of SEQ ID NO: 82), SEQ ID NO: 4 (nucleic acid sequence of SEQ ID NO: 85), and SEQ ID NO: 8 (nucleic acid sequence of SEQ ID NO: 89), can achieve greater than 30% indels via the NHEJ pathway on a target gene when fused to a DNA binding domain of a TALE, RNBD, and MAP-NBD.

A non-naturally occurring fusion protein of the disclosure, e.g., any one of SEQ ID NO: 1-SEQ ID NO: 81 (or any one of nucleic acid sequences of SEQ ID NO: 82-SEQ ID NO: 162) fused to a DNA binding domain, can comprise a repeat unit. A repeat unit can be from a wild-type DNA-binding domain (Ralstonia solanacearum, Xanthomonas spp., Legionella quateirensis, Burkholderia, Paraburkholderia, or Francisella) or a modified repeat unit enhanced for specific recognition of a particular nucleic acid base. A modified repeat unit can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more mutations that can enhance the repeat module for specific recognition of a particular nucleic acid base. In some embodiments, a modified repeat unit is modified at amino acid position 2, 3, 4, 11, 12, 13, 21, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, or 35. In some embodiments, a modified repeat unit is modified at amino acid positions 12 or 13.

As described in further detail below, a non-naturally occurring fusion protein of the disclosure, e.g., anyone of SEQ ID NO: 1-SEQ ID NO: 81 (or any one of nucleic acid sequences of SEQ ID NO: 82-SEQ ID NO: 162) fused to a plurality of repeat units (e.g., derived from Ralstonia solanacearum, Xanthomonas spp., Legionella quateirensis, Burkholderia, Paraburkholderia, or Francisella), can further comprise a C-terminal truncation, which can served as a linker between the DNA binding domain and the nuclease.

A non-naturally occurring fusion protein of the disclosure, e.g., anyone of SEQ ID NO: 1-SEQ ID NO: 81 (or any one of nucleic acid sequences of SEQ ID NO: 82-SEQ ID NO: 162) fused to a DNA binding domain, can further comprise an N-terminal cap as described in further detail below. An N-terminal cap can be a polypeptide portion flanking the DNA-binding repeat module. An N-terminal cap can be any length and can comprise from 0 to 136 amino acid residues in length. An N-terminal cap can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acid residues in length. In some embodiments, an N-terminal cap can modulate structural stability of the DNA-binding repeat units. In some embodiments, an N-terminal cap can modulate nonspecific interactions. In some cases, an N-terminal cap can decrease nonspecific interaction. In some cases, an N-terminal cap can reduce off-target effect. As used here, off-target effect refers to the interaction of a genome editing complex with a sequence that is not the target binding site of interest. An N-terminal cap can further comprise a wild-type N-terminal cap sequence of a protein from Ralstonia solanacearum, Xanthomonas spp., Legionella quateirensis, Burkholderia, Paraburkholderia, or Francisella or can comprise a modified N-terminal cap sequence.

In some embodiments, a DNA binding domain comprises at least one repeat unit having a repeat variable diresidue (RVD), which contacts a target nucleic acid base. In some embodiments, a DNA binding domain comprises more than one repeat unit, each having an RVD, which contacts a target nucleic acid base. In some embodiments, the DNA binding domain comprises 1 to 50 RVDs. In some embodiments, the DNA binding domain components of the fusion proteins can be at least 14 RVDs, at least 15 RVDs, at least 16 RVDs, at least 17 RVDs, at least 18 RVDs, at least 19 RVDs, at least 20 RVDs in length, or at least 21 RVDs in length. In some embodiments, the DNA binding domains can be 16 to 21 RVDs in length.

In some embodiments, any one of the DNA binding domains described herein can bind to a region of interest of any gene. For example, the DNA binding domains described herein can bind upstream of the promoter region, upstream of the gene transcription start site, or downstream of the transcription start site. In certain embodiments, the DNA binding domain binding region is no farther than 50 base pairs downstream of the transcription start site. In some embodiments, the DNA binding domain is designed to bind in proximity to the transcription start site (TSS). In other embodiments, the TALE can be designed to bind in the 5′ UTR region.

A DNA binding domain described herein can comprise between 1 to 50 repeat units. A DNA binding domain described herein can comprise between 5 and 45, between 8 to 45, between 10 to 40, between 12 to 35, between 15 to 30, between 20 to 30, between 8 to 40, between 8 to 35, between 8 to 30, between 10 to 35, between 10 to 30, between 10 to 25, between 10 to 20, or between 15 to 25 repeat units.

A DNA binding domain described herein can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, or more repeat units. A DNA binding domain described herein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, or 50 repeat units. A DNA binding domain described herein can comprise 5 repeat units. A DNA binding domain described herein can comprise 10 repeat units. A DNA binding domain described herein can comprise 11 repeat units. A DNA binding domain described herein can comprise 12 repeat units, or another suitable number.

A repeat unit of a DNA binding domain can be 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 37, 38, 39 or 40 residues in length.

In some embodiments, the effector can be a protein secreted from Xanthomonas or Ralstonia bacteria upon plant infection. In some embodiments, the effector can be a protein that is a mutated form of, or otherwise derived from, a protein secreted from Xanthomonas or Ralstonia bacteria. The effector can further comprise a DNA-binding module which includes a variable number of about 33-35 amino acid residue repeat units. Each amino acid repeat unit recognizes one base pair through two adjacent amino acids (e.g., at amino acid positions 12 and 13 of the repeat unit). As such, amino acid positions 12 and 13 of the repeat unit can also be referred to as repeat variable diresidue (RVD).

Linkers

A nuclease, e.g., anyone of SEQ ID NO: 1-SEQ ID NO: 81 (or any one of nucleic acid sequences of SEQ ID NO: 82-SEQ ID NO: 162) fused to a DNA binding domain (e.g., an RNBD, a MAP-NBD, a TALE), can further include a linker connecting SEQ ID NO: 1-SEQ ID NO: 81 (or any one of nucleic acid sequences of SEQ ID NO: 82-SEQ ID NO: 162) to the DNA binding domain. A linker used herein can be a short flexible linker comprising 0 base pairs, 3 to 6 base pairs, 6 to 12 base pairs, 12 to 15 base pairs, 15 to 21 base pairs, 21 to 24 base pairs, 24 to 30 base pairs, 30 to 36 base pairs, 36 to 42 base pairs, 42 to 48 base pairs, or 1 to 48 base pairs. The nucleic acid sequence of the linker can encode for an amino acid sequence comprising 0 residues, 1-3 residues, 4-7 residues, 8-10 residues, 10-12 residues, 12-15 residues, or 1-15 residues. Linkers can include, but are not limited to, residues such as glycine, methionine, aspartic acid, alanine, lysine, serine, leucine, threonine, tryptophan, or any combination thereof.

When linking a repressor domain to an RNBD, MAP-NBD, or TALE, the linker can have a nucleic acid sequence of GGCGGTGGCGGAGGGATGGATGCTAAGTCACTAACTGCCTGGTCC (SEQ ID NO: 165) and an amino acid sequence of GGGGGMDAKSLTAWS (SEQ ID NO: 166).

A nuclease, e.g., anyone of SEQ ID NO: 1-SEQ ID NO: 81 (or any one of nucleic acid sequences of SEQ ID NO: 82-SEQ ID NO: 162) can be connected to a DNA binding domain via a linker, a linker can be between 1 to 70 amino acid residues in length. A linker can be from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, from 5 to 20, from 5 to 15, from 10 to 40, from 10 to 35, from 10 to 30, from 10 to 25, from 10 to 20, from 12 to 40, from 12 to 35, from 12 to 30, from 12 to 25, from 12 to 20, from 14 to 40, from 14 to 35, from 14 to 30, from 14 to 25, from 14 to 20, from 14 to 16, from 15 to 40, from 15 to 35, from 15 to 30, from 15 to 25, from 15 to 20, from 15 to 18, from 18 to 40, from 18 to 35, from 18 to 30, from 18 to 25, from 18 to 24, from 20 to 40, from 20 to 35, from 20 to 30, from 25 to 30, from 25 to 70, from 30 to 70, from 5 to 70, from 35 to 70, from 40 to 70, from 45 to 70, from 50 to 70, from 55 to 70, from 60 to 70, or from 65 to 70 amino acid residues in length.

A linker for linking a nuclease, e.g., anyone of SEQ ID NO: 1-SEQ ID NO: 81 (or any one of nucleic acid sequences of SEQ ID NO: 82-SEQ ID NO: 162) to a DNA binding domain can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acid residues in length.

In some embodiments, the linker can be the N-terminus of a naturally occurring Ralstonia solanacearum-derived protein, Xanthomonas spp.-derived protein, or animal pathogen-derived protein, wherein any functional domain disclosed herein is fused to the N-terminus of the engineered DNA binding domain. In some embodiments, the linker comprising the N-terminus can comprise the full length naturally occurring N-terminus of a naturally occurring Ralstonia solanacearum-derived protein, Xanthomonas spp.-derived protein, or animal pathogen-derived protein, or a truncation of the naturally occurring N-terminus, such as amino acid residues at positions 1 to 137 of the naturally occurring Ralstonia solanacearum-derived protein N-terminus (e.g., SEQ ID NO: 264), positions 1 (H) to 115 (S) of the naturally occurring Ralstonia solanacearum-derived protein N-terminus (SEQ ID NO: 320), positions 1 (N) to 115 (S) of the naturally occurring Xanthomonas spp.-derived protein N-terminus (SEQ ID NO: 321), or positions 1 (G) to 115 (K) of the naturally occurring Legionella quateirensis-derived protein N-terminus (SEQ ID NO: 322). In some embodiments, the linker can comprise amino acid residues at positions 1 to 120 of the naturally occurring Ralstonia solanacearum-derived protein (SEQ ID NO: 303), Xanthomonas spp.-derived protein (SEQ ID NO: 301), or Legionella quateirensis-derived protein (SEQ ID N): 304). In some embodiments, the linker can comprise the naturally occurring N-terminus of Ralstonia solanacearum truncated to any length. For example, the naturally occurring N-terminus of Ralstonia solanacearum can be truncated to amino acid residues at positions 1 to 120, 1 to 115, 1 to 50, 1 to 70, 1 to 100, 1 to 120, 1 to 130, 10 to 40, 60 to 100, or 100 to 120 and used at the N-terminus of the engineered DNA binding domain as a linker to a nuclease or a repressor.

In other embodiments, the linker can be the C-terminus of a naturally occurring Ralstonia solanacearum-derived protein, Xanthomonas spp.-derived protein, or animal pathogen-derived protein, wherein any functional domain disclosed herein is fused to the C-terminus of the engineered DNA binding domain. In some embodiments, the linker comprising the C-terminus can comprise the full length naturally occurring C-terminus of a naturally occurring Ralstonia solanacearum-derived protein, Xanthomonas spp.-derived protein, or animal pathogen-derived protein, or a truncation of the naturally occurring C-terminus, such as positions 1 to 63 of the naturally occurring Ralstonia solanacearum-derived protein (SEQ ID NO: 266), Xanthomonas spp.-derived protein (SEQ ID NO: 298), or Legionella quateirensis-derived protein (SEQ ID NO: 306). In some embodiments, the naturally occurring C-terminus of Ralstonia solanacearum-derived protein, Xanthomonas spp.-derived protein, or Legionella quateirensis-derived protein can be truncated to any length and used at the C-terminus of the engineered DNA binding domain and used as a linker to a nuclease or repressor. For example, the naturally occurring C-terminus of Ralstonia solanacearum-derived protein, Xanthomonas spp.-derived protein, or Legionella quateirensis-derived protein can be truncated to amino acid residues at positions 1 to 63, 1 to 50, 1 to 70, 1 to 100, 1 to 120, 1 to 130, 10 to 40, 60 to 100, or 100 to 120 and used at the C-terminus of the engineered DNA binding domain.

Linkers Comprising Recognition Sites

In some embodiments, the present disclosure provides DNA binding domains (e.g., RNBDs, MAP-NBDs, TALEs) with gapped repeat units for use as gene editing complexes. A DNA binding domain (e.g., RNBDs, MAP-NBDs, TALEs) with gapped repeat can comprise of a plurality of repeat units in which each repeat unit of the plurality of repeat units is separated from a neighboring repeat unit by a linker. This linker can comprise a recognition site for additional functionality and activity. For example, the linker can comprise a recognition site for a small molecule. As another example, the linker can serve as a recognition site for a protease. In yet another example, the linker can serve as a recognition site for a kinase. In other embodiments, the recognition site can serve as a localization signal.

Each repeat unit of a DNA binding domain (e.g., RNBDs, MAP-NBDs, TALEs) comprises a secondary structure in which the RVD interfaces with and binds to a target nucleic acid base on double stranded DNA, while the remainder of the repeat unit protrudes from the surface of the DNA. Thus, the linkers comprising a recognition site between each repeat unit are removed from the surface of the DNA and are solvent accessible. In some embodiments, these solvent accessible linkers comprising recognition sites can have extra activity while mediating gene editing. In some embodiments, the at least one repeat unit comprises 1-20 additional amino acid residues at the C-terminus. In some aspects, the at least repeat unit of the plurality of repeat units is separated from a neighboring repeat unit by a linker. In further aspects, the linker comprises a recognition site. In some aspects, the recognition site is for a small molecule, a protease, or a kinase. In some aspects, the recognition site serves as a localization signal. In some aspects, the plurality of repeat units comprises 3 to 60 repeat units.

Examples of a left and a right DNA binding domain comprising repeat units derived from Xanthomonas spp. are shown below in TABLE 7 for AAVS1 and GA7. “X,” shown in bold and underlining, represents a linker comprising a recognition site and can comprise 1-40 amino acid residues. An amino acid residue of the linker can comprise a glycine, an alanine, a threonine, or a histidine.

TABLE 7 Exemplary Left or Right Gapped DNA Binding Domains SEQ ID NO Construct Sequence 307 AAVS1_Left LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGG KQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLC QDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIA SNGGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGGKQALETVQRL LPVLCQDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQ VVAIASNIGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGGKQALET VQRLLPVLCQDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X L TPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGGK QALETVQRLLPVLCQDHG X LTPDQVVAIASNIGGKQALETVQRLLPVLCQD HG X LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNI GGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNHGGKQALETVQRLLPV LCQDHGXLTPDQVVAIASNGGG 308 AAVS1_Right LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNGGG KQALETVQRLLPVLCQDHG X LTPDQVVAIASNGGGKQALETVQRLLPVLC QDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIA SNGGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNHGGKQALETVQRL LPVLCQDHG X LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG X LTPDQ VVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNIGGKQALET VQRLLPVLCQDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X L TPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNIGGK QALETVQRLLPVLCQDHG X LTPDQVVAIASNIGGKQALETVQRLLPVLCQD HG X LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASH DGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGGKQALETVQRLLP VLCQDHG X LTPDQVVAIASNGGGKQALESIVAQLSRPDPALA 309 GA7.2 Left LTPDQVVAIASNHGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGG KQALETVQRLLPVLCQDHG X LTPDQVVAIASNGGGKQALETVQRLLPVLC QDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIA SNIGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNHGGKQALETVQRL LPVLCQDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQ VVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGGKQALE TVQRLLPVLCQDHG X LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNHGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGG KQALETVQRLLPVLCQDHG X LTPDQVVAIASNGGGKQALETVQRLLPVLC QDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIA SNIGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNHGGKQALETVQRL LPVLCQDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQ VVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNGGGK 310 GA7.2 Right LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGG KQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLC QDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIA SHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNGGGKQALETVQRL LPVLCQDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQ VVAIASNGGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGGKQALE TVQRLLPVLCQDHG X LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNGGG KQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGGKQALETVQRLLPVLC QDHG X LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG X LTPDQVVAIA SHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASNGGGKQALETVQRL LPVLCQDHG X LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGXLTPDQ VVAIASHDGGKQALETVQRLLPVLCQDHG X LTPDQVVAIASHDGGKQALE TVQRLLPVLCQDHG X LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG X LTPDQVVASASNGGGKQALESIVAQLSRPDPALA

Tunable Repeat Units

In some embodiments, the present disclosure provides DNA binding domains (e.g., RNBDs, MAP-NBDs, TALEs) with expanded repeat units. For example, a DNA binding domain (e.g., RNBDs, MAP-NBDs, TALEs) comprises a plurality of repeat units in which each repeat unit is usually 33-35 amino acid residues in length. The present disclosure provides repeat units, which are greater than 35 amino acid residues in length. In some embodiments, the present disclosure provides repeat units, which are greater than 39 amino acid residues in length. In some embodiments, the present disclosure provides repeat units which are 35 to 40, 39 to 40, 35 to 45, 39 to 45, 35 to 50, 39 to 50, 35 to 50, 35 to 60, 39 to 60, 35 to 70, 39 to 70, 35 to 79, or 39 to 79 amino acid residues long.

In other embodiments, the present disclosure provides DNA binding domains (e.g., RNBDs, MAP-NBDs, TALEs) with contracted repeat units. For example, the present disclosure provides repeat units, which are less than 32 amino acid residues in length. In some embodiments, the present disclosure provides repeat units, which are 15 to 32, 16 to 32, 17 to 32, 18 to 32, 19 to 32, 20 to 32, 21 to 32, 22 to 32, 23 to 32, 24 to 32, 25 to 32, 26 to 32, 27 to 32, 28 to 32, 29 to 32, 30 to 32, or 31 to 32 amino acid residues in length.

In some embodiments, said expanded repeat units can be tuned to modulate binding of each repeat unit to its target nucleic acid, resulting in the ability to overall modulate binding of the DNA binding domain (e.g., RNBDs, MAP-NBDs, TALEs) to a target gene of interest. For example, expanding repeat units can improve binding affinity of the repeat unit to its target nucleic acid base and thereby increase binding affinity of the DNA binding domain (e.g., RNBDs, MAP-NBDs, TALEs) to a target gene. In other embodiments, contracting repeat units can improve binding affinity of the repeat unit to its target nucleic acid base and thereby increase binding affinity of the DNA binding domain (e.g., RNBDs, MAP-NBDs, TALEs) for a target gene.

Functional Domains

An RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), can be linked to a functional domain. The functional domain can provide different types activity, such as genome editing, gene regulation (e.g., activation or repression), or visualization of a genomic locus via imaging.

A. Genome Editing Domains

For example, an RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), can be linked to a nuclease, wherein the RNBD provides specificity and targeting and the nuclease provides genome editing functionality. In some embodiments, the nuclease can be a cleavage domain, which dimerizes with another copy of the same cleavage domain to form an active full domain capable of cleaving DNA. In other embodiments, the nuclease can be a cleavage domain, which is capable of cleaving DNA without needing to dimerize. For example, a nuclease comprising a cleavage domain can be an endonuclease, such as FokI or Bfil. In some embodiments, two cleavage domains (e.g., FokI or Bfil) can be fused together to form a fully functional single cleavage domain. When cleavage domains are used as the nuclease, two RNBDs can be engineered, the first RNBD binding to a top strand of a target nucleic acid sequence and comprising a first FokI cleavage domain and a second RNBD binding to a bottom strand of a target nucleic acid sequence and comprising a second FokI cleavage domain.

In some embodiments, a fully functional cleavage domain, capable of cleaving DNA without needing to dimerize include meganucleases, also referred to as homing endonucleases. For example, a meganuclease can include I-Anil or I-OnuI. In some embodiments, the nuclease can be a type IIS restriction enzyme, such as FokI or Bfil.

A nuclease domain fused to an RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), can be an endonuclease or an exonuclease. An endonuclease can include restriction endonucleases and homing endonucleases. An endonuclease can also include S1 Nuclease, mung bean nuclease, pancreatic DNase I, micrococcal nuclease, or yeast HO endonuclease. An exonuclease can include a 3′-5′ exonuclease or a 5′-3′ exonuclease. An exonuclease can also include a DNA exonuclease or an RNA exonuclease. Examples of exonuclease includes exonucleases I, II, III, IV, V, and VIII; DNA polymerase I, RNA exonuclease 2, and the like.

A nuclease domain fused to an RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), can be a restriction endonuclease (or restriction enzyme). In some instances, a restriction enzyme cleaves DNA at a site removed from the recognition site and has a separate binding and cleavage domains. In some instances, such restriction enzyme is a Type IIS restriction enzyme.

A nuclease domain fused to an RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), can be a Type IIS nuclease. A Type IIS nuclease can be FokI or Bfil. In some cases, a nuclease domain fused to an RNBD (e.g., Ralstonia solanacearum-derived) is FokI. In other cases, a nuclease domain fused to an RNBD (e.g., Ralstonia solanacearum-derived) is Bfil.

FokI can be a wild-type FokI or can comprise one or more mutations. In some cases, FokI can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. A mutation can enhance cleavage efficiency. A mutation can abolish cleavage activity. In some cases, a mutation can modulate homodimerization. For example, FokI can have a mutation at one or more amino acid residue positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 to modulate homodimerization.

In some instances, a FokI cleavage domain is, for example, as described in Kim et al. “Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain,” PNAS 93: 1156-1160 (1996), which is incorporated herein by reference in its entirety. In some cases, a FokI cleavage domain described herein has a sequence as follows: QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLG GSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWW KVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEE VRRKFNNGEINF (SEQ ID NO: 163). In other instances, a FokI cleavage domain described herein is a FokI, for example, as described in U.S. Pat. No. 8,586,526, which is incorporated herein by reference in its entirety.

An RNBD (e.g., Ralstonia solanacearum-derived) can be linked to a functional group that modifies DNA nucleotides, for example an adenosine deaminase.

In some embodiments, an RNBD (e.g., Ralstonia solanacearum-derived) can be linked to any nuclease as set forth in TABLE 6 showing exemplary amino acid sequences (SEQ ID NO: 1-SEQ ID NO: 81) of endonucleases for genome editing and the corresponding back-translated nucleic acid sequences (SEQ ID NO: 82-SEQ ID NO: 162) of the endonucleases.

For purposes of gene editing, a first DNA binding domain (e.g., of a TALE, RNBD, or MAP-NBD) linked to a cleavage domain and a second DNA binding domain (e.g., of a TALE, RNBD, or MAP-NBD) linked to a cleavage domain can be provided. The first DNA binding domain (e.g., of a TALE, RNBD, or MAP-NBD) linked to a cleavage domain can recognize a top strand of double stranded DNA and bind to said region of double stranded DNA. The second DNA binding domain (e.g., of a TALE, RNBD, or MAP-NBD) linked to a cleavage domain can recognize a separate, non-overlapping bottom strand of double stranded DNA and bind to said region of double stranded DNA. The target nucleic acid sequence on the bottom strand can have its complementary nucleic acid sequence in the top strand positioned 10 to 20 nucleotides towards the 3′ end from the first region. In some embodiments this stretch of 10 to 20 nucleotides can be referred to as the spacer region. In some embodiments, this first DNA binding domain (e.g., of a TALE, RNBD, or MAP-NBD) linked to a cleavage domain and the second DNA binding domain (e.g., of a TALE, RNBD, or MAP-NBD) linked to a cleavage domain both bind at a target site, allowing for dimerization of the two cleavage domains in the spacer region and allowing for catalytic activity and cleaving of the target DNA.

a. Potency and Specificity of Genome Editing

In some embodiments, the efficiency of genome editing with a genome editing complex of the present disclosure (e.g., any one of an RNBD, MAP-NBD, or TALE fused to any nuclease disclosed herein) can be determined. Specifically, the potency and specificity of the genome editing complex can indicate whether a particular modular nucleic acid binding domain fused to a nuclease provides efficient editing. Potency can be defined as the percent indels (insertions/deletions) that are generated via the non-homologous end joining (NHEJ) pathway at a target site after administering a modular nucleic acid binding domain fused to a nuclease to a subject. A modular nucleic acid binding domain can have a potency of greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 92%, greater than 95%, greater than 97%, or greater than 99%. A modular nucleic acid binding domain can have a potency of from 50% to 100%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

Specificity can be defined as a specificity ratio, wherein the ratio is the percent indels at a target site of interest over the percent indels at the top-ranked off-target site for a particular genome editing complex (e.g., any DNA binding domain linked to a nuclease described herein) of interest. A high specificity ratio would indicate that a modular nucleic acid binding domain fused to a nuclease edits primarily at the desired target site and exhibits fewer instances of undesirable, off-target editing. A low specificity ratio would indicate that a modular nucleic acid binding domain fused to a nuclease does not edit efficiently at the desired target site and/or can indicate that the modular nucleic acid binding domain fused to a nuclease exhibits high off-target activity. A modular nucleic acid binding domain can have a specificity ratio for the target site of at least 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 92:1, 95:1, 97:1, 99:1, 50:2, 55:2, 60:2, 65:2, 70:2, 75:2, 80:2, 85:2, 90:2, 92:2, 95:2, 97:2, 99:2, 50:3, 55:3, 60:3, 65:3, 70:3, 75:3, 80:3, 85:3, 90:3, 92:3, 95:3, 97:3, 99:3, 50:4, 55:4, 60:4, 65:4, 70:4, 75:4, 80:4, 85:4, 90:4, 92:4, 95:4, 97:4, 99:4, 50:5, 55:5, 60:5, 65:5, 70:5, 75:5, 80:5, 85:5, 90:5, 92:5, 95:5, 97:5, or 99:5. A modular nucleic acid binding domain can have a specificity ratio for the target site from 50:1 to 100:1, 99:5 to 50:1, or 99:5 to 100:1. Percent indels can be measured via deep sequencing techniques.

In some embodiments, the present disclosure provides a polypeptide comprising a modular nucleic acid binding domain comprising a potency for a target site greater than 65% and a specificity ratio for the target site of at least 50:1; and a functional domain; wherein: the modular nucleic acid binding domain comprises a plurality of repeat units; at least one repeat unit of the plurality of repeat units comprises a binding region configured to bind to a target nucleic acid base within the target site; the potency comprises indel percentage at the target site, and wherein the specificity ratio comprises indel percentage at the target site over indel percentage at a top-ranked off-target site of the polypeptide. Indel percentage can be measured by deep sequencing.

The top-ranked off-target site for a polypeptide (e.g., a modular nucleic acid binding domain linked to a cleavage domain) can be determined using the predicted report of genome-wide nuclease off-target sites (PROGNOS) ranking algorithms as described in Fine et al. (Nucleic Acids Res. 2014 April; 42(6):e42. doi: 10.1093/nar/gkt1326. Epub 2013 Dec. 30.). As described in Fine et al, the PROGNOS algorithm TALEN v2.0 can use the DNA target sequence as input; prior construction and experimental characterization of the specific nucleases are not necessary. Based on the differences between the sequence of a potential off-target site in the genome and the intended target sequence, the algorithm can generate a score that is used to rank potential off-target sites. If two (or more) potential off-target sites have equal scores, they can be further ranked by the type of genomic region annotated for each site with the following order: Exon>Promoter>Intron>Intergenic. A final ranking by chromosomal location can be employed as a tie-breaker to ensure consistency in the ranking order. Thus, a score can be generated for each potential off-target site.

B. Regulatory Domains

As another example, an RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), can be linked to a gene regulating domain. A gene regulation domain can be an activator or a repressor. For example, an RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), can be linked to an activation domain, such as VP16, VP64, p65, p300 catalytic domain, TET1 catalytic domain, TDG, Ldb1 self-associated domain, SAM activator (VP64, p65, HSF1), or VPR (VP64, p65, Rta). Alternatively, an RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), can be linked to a repressor, such as KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID MBD2, MBD3, Rb, or MeCP2.

In some embodiments, an RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), can be linked to a DNA modifying protein, such as DNMT3a. An RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), can be linked to a chromatin-modifying protein, such as lysine-specific histone demethylase 1 (LSD1). An RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), can be linked to a protein that is capable of recruiting other proteins, such as KRAB. The DNA modifying protein (e.g., DNMT3a) and proteins capable of recruiting other proteins (e.g., KRAB) can serve as repressors of transcription. Thus, RNBDs (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), linked to a DNA modifying protein (e.g., DNMT3a) or a domain capable of recruiting other proteins (e.g., KRAB, a domain found in transcriptional repressors, such as Kox1) can provide gene repression functionality, can serve as transcription factors, wherein the RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), provides specificity and targeting and the DNA modifying protein and the protein capable of recruiting other proteins provides gene repression functionality, which can be referred to as a TALE-transcription factor (TALE-TF), RNBD-transcription factor (RNBD-TF), or MAP-NBD-transcription factor (MAP-NBD-TF).

In some embodiments, expression of the target gene can be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% by using a DNA binding domain fused to a repression domain (e.g., an RNBD-TF, a MAP-NBD-TF, or TALE-TF) of the present disclosure as compared to non-treated cells. In some embodiments, expression of the target gene can be reduced by 5% to 10%, 10% to 15%, 15% to 20%, 20%, to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% by using an RNBD-TF, a MAP-NBD-TF, or TALE-TF of the present disclosure as compared to non-treated cells. In some embodiments, expression of the checkpoint gene can be reduced by over 90% by using an RNBD-TF, a MAP-NBD-TF, or TALE-TF of the present disclosure as compared to non-treated cells.

In some embodiments, repression of the target gene with a DNA binding domain fused to a repression domain (e.g., an RNBD-TF, a MAP-NBD-TF, or TALE-TF) of the present disclosure and subsequent reduced expression of the target gene can last for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, or at least 28 days. In some embodiments, repression of the target gene with an RNBD-TF, a MAP-NBD-TF, or TALE-TF of the present disclosure and subsequent reduced expression of the target gene can last for 1 days to 3 days, 3 days to 5 days, 5 days to 7 days, 7 days to 9 days, 9 days to 11 days, 11 days to 13 days, 13 days to 15 days, 15 days to 17 days, 17 days to 19 days, 19 days to 21 days, 21 days to 23 days, 23 days to 25 days, or 25 days to 28 days.

In various aspects, the present disclosure provides a method of identifying a target binding site in a target gene of a cell, the method comprising: (a) contacting a cell with an engineered genomic regulatory complex comprising a DNA binding domain, a repressor domain, and a linker; (b) measuring expression of the target gene; and (c) determining expression of the target gene is repressed by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% for at least 3 days, wherein the target gene is selected from: a checkpoint gene and a T cell surface receptor.

In some aspects, expression of the target gene is repressed in at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of a plurality of the cells. In some aspects, the engineered genomic regulatory complex is undetectable after at least 3 days. In some aspects, determining the engineered genomic regulatory complex is undetectable is measured by qPCR, imaging of a FLAG-tag, or a combination thereof. In some aspects, the measuring expression of the target gene comprises flow cytometry quantification of expression of the target gene.

In some embodiments, repression of the target gene with a DNA binding domain fused to a repression domain (e.g., an RNBD-TF, a MAP-NBD-TF, or TALE-TF) of the present disclosure can last even after the DNA binding domain-gene regulator becomes undetectable. The DNA binding domain fused to a repression domain (e.g., an RNBD-TF, a MAP-NBD-TF, or TALE-TF) can become undetectable after at least 3 days. In some embodiments, the DNA binding domain fused to a repression domain (e.g., an RNBD-TF, a MAP-NBD-TF, or TALE-TF) can become undetectable after at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks. In some embodiments, qPCR or imaging via the FLAG-tag can be used to confirm that the DNA binding domain fused to a repression domain (e.g., an RNBD-TF, a MAP-NBD-TF, or TALE-TF) is no longer detectable.

C. Imaging Moieties

An RNBD (e.g., Ralstonia solanacearum-derived), or another binding domain (e.g., MAP-NBD or TALE), can be linked to a fluorophore, such as Hydroxycoumarin, methoxycoumarin, Alexa fluor, aminocoumarin, Cy2, FAM, Alexa fluor 488, Fluorescein FITC, Alexa fluor 430, Alexa fluor 532, HEX, Cy3, TRITC, Alexa fluor 546, Alexa fluor 555, R-phycoerythrin (PE), Rhodamine Red-X, Tamara, Cy3.5, Rox, Alexa fluor 568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633, Allophycocyanin, Alexa fluor 633, Cy5, Alexa fluor 660, Cy5.5, TruRed, Alexa fluor 680, Cy7, GFP, or mCHERRY. An RNBD (e.g., Ralstonia solanacearum-derived) can be linked to a biotinylation reagent.

Genes and Indications of Interest

In some embodiments, genome editing can be performed by fusing a nuclease of the present disclosure with a DNA binding domain for a particular genomic locus of interest. Genetic modification can involve introducing a functional gene for therapeutic purposes, knocking out a gene for therapeutic gene, or engineering a cell ex vivo (e.g., HSCs or CAR T cells) to be administered back into a subject in need thereof. For example, the genome editing complex can have a target site within PDCD1, CTLA4, LAGS, TET2, BTLA, HAVCR2, CCR5, CXCR4, TRA, TRB, B2M, albumin, HBB, HBA1, TTR, NR3C1, CD52, erythroid specific enhancer of the BCL11A gene, CBLB, TGFBR1, SERPINA1, HBV genomic DNA in infected cells, CEP290, DMD, CFTR, IL2RG, CS-1, or any combination thereof. In some embodiments, a genome editing complex can cleave double stranded DNA at a target site in order to insert a chimeric antigen receptor (CAR), alpha-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), or Factor 9 (F9). Cells, such as hematopoietic stem cells (HSCs) and T cells, can be engineered ex vivo with the genome editing complex. Alternatively, genome editing complexes can be directly administered to a subject in need thereof.

The subject receiving treatment can be suffering from a disease such as transthyretin amyloidosis (ATTR), HIV, glioblastoma multiforme, cancer, acute lymphoblastic leukemia, acute myeloid leukemia, beta-thalassemia, sickle cell disease, MPSI, MPSII, Hemophilia B, multiple myeloma, melanoma, sarcoma, Leber congenital amaurosis (LCA10), CD19 malignancies, BCMA-related malignancies, duchenne muscular dystrophy (DMD), cystic fibrosis, alpha-1 antitrypsin deficiency, X-linked severe combined immunodeficiency (X-SCID), or Hepatitis B.

Samples for Analysis

In some aspects, described herein include methods of modifying the genetic material of a target cell utilizing an RNBD described herein. A sample described herein may be a fresh sample. The sample may be a live sample.

The sample may be a cell sample. The cell sample may be obtained from the cells or tissue of an animal. The animal cell may comprise a cell from an invertebrate, fish, amphibian, reptile, or mammal. The mammalian cell may be obtained from a primate, ape, equine, bovine, porcine, canine, feline, or rodent. The mammal may be a primate, ape, dog, cat, rabbit, ferret, or the like. The rodent may be a mouse, rat, hamster, gerbil, hamster, chinchilla, or guinea pig. The bird cell may be from a canary, parakeet, or parrot. The reptile cell may be from a turtle, lizard, or snake. The fish cell may be from a tropical fish. For example, the fish cell may be from a zebrafish (such as Danio rerio). The amphibian cell may be from a frog. An invertebrate cell may be from an insect, arthropod, marine invertebrate, or worm. The worm cell may be from a nematode (such as Caenorhabditis elegans). The arthropod cell may be from a tarantula or hermit crab.

The cell sample may be obtained from a mammalian cell. For example, the mammalian cell may be an epithelial cell, connective tissue cell, hormone secreting cell, a nerve cell, a skeletal muscle cell, a blood cell, an immune system cell, or a stem cell. A cell may be a fresh cell, live cell, fixed cell, intact cell, or cell lysate. Cell samples can be any primary cell, such as a hematopoetic stem cell (HSCs) or naïve or stimulated T cells (e.g., CD4+ T cells).

Cell samples may be cells derived from a cell line, such as an immortalized cell line. Exemplary cell lines include, but are not limited to, 293A cell line, 293FT cell line, 293F cell line, 293 H cell line, HEK 293 cell line, CHO DG44 cell line, CHO-S cell line, CHO-K1 cell line, Expi293F™ cell line, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™-CV-1 cell line, Flp-In™-Jurkat cell line, FreeStyle™ 293-F cell line, FreeStyle™ CHO-S cell line, GripTite™ 293 MSR cell line, GS-CHO cell line, HepaRG™ cell line, T-REx™ Jurkat cell line, Per.C6 cell line, T-REx™-293 cell line, T-REx™-CHO cell line, T-REx™-HeLa cell line, NC-HIMT cell line, PC12 cell line, A549 cells, and K562 cells.

In some embodiments, an RNBD of the present disclosure can be used to modify a target cell. The target cell can itself be unmodified or modified. For example, an unmodified cell can be edited with an RNBD of the present disclosure to introduce an insertion, deletion, or mutation in its genome. In some embodiments, a modified cell already having a mutation can be repaired with an RNBD of the present disclosure.

In some instances, a target cell is a cell comprising one or more single nucleotide polymorphism (SNP). In some instances, an RNBD-nuclease described herein is designed to target and edit a target cell comprising a SNP.

In some cases, a target cell is a cell that does not contain a modification. For example, a target cell can comprise a genome without genetic defect (e.g., without genetic mutation) and an RNBD-nuclease described herein can be used to introduce a modification (e.g., a mutation) within the genome.

The cell sample may be obtained from cells of a primate. The primate may be a human, or a non-human primate. The cell sample may be obtained from a human. For example, the cell sample may comprise cells obtained from blood, urine, stool, saliva, lymph fluid, cerebrospinal fluid, synovial fluid, cystic fluid, ascites, pleural effusion, amniotic fluid, chorionic villus sample, vaginal fluid, interstitial fluid, buccal swab sample, sputum, bronchial lavage, Pap smear sample, or ocular fluid. The cell sample may comprise cells obtained from a blood sample, an aspirate sample, or a smear sample.

The cell sample may be a circulating tumor cell sample. A circulating tumor cell sample may comprise lymphoma cells, fetal cells, apoptotic cells, epithelia cells, endothelial cells, stem cells, progenitor cells, mesenchymal cells, osteoblast cells, osteocytes, hematopoietic stem cells (HSC) (e.g., a CD34+HSC), foam cells, adipose cells, transcervical cells, circulating cardiocytes, circulating fibrocytes, circulating cancer stem cells, circulating myocytes, circulating cells from a kidney, circulating cells from a gastrointestinal tract, circulating cells from a lung, circulating cells from reproductive organs, circulating cells from a central nervous system, circulating hepatic cells, circulating cells from a spleen, circulating cells from a thymus, circulating cells from a thyroid, circulating cells from an endocrine gland, circulating cells from a parathyroid, circulating cells from a pituitary, circulating cells from an adrenal gland, circulating cells from islets of Langerhans, circulating cells from a pancreas, circulating cells from a hypothalamus, circulating cells from prostate tissues, circulating cells from breast tissues, circulating cells from circulating retinal cells, circulating ophthalmic cells, circulating auditory cells, circulating epidermal cells, circulating cells from the urinary tract, or combinations thereof.

The cell can be a T cell. For example, in some embodiments, the T cell can be an engineered T cell transduced to express a chimeric antigen receptor (CAR). The CAR T cell can be engineered to bind to BCMA, CD19, CD22, WT1, L1 CAM, MUC16, ROR1, or LeY.

A cell sample may be a peripheral blood mononuclear cell sample.

A cell sample may comprise cancerous cells. The cancerous cells may form a cancer which may be a solid tumor or a hematologic malignancy. The cancerous cell sample may comprise cells obtained from a solid tumor. The solid tumor may include a sarcoma or a carcinoma. Exemplary sarcoma cell sample may include, but are not limited to, cell sample obtained from alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, clear cell sarcoma of soft tissue, dedifferentiated liposarcoma, desmoid, desmoplastic small round cell tumor, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma, esthesioneuroblastoma, Ewing sarcoma, extrarenal rhabdoid tumor, extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma, infantile fibrosarcoma, inflammatory myofibroblastic tumor, Kaposi sarcoma, leiomyosarcoma of bone, liposarcoma, liposarcoma of bone, malignant fibrous histiocytoma (MFH), malignant fibrous histiocytoma (MFH) of bone, malignant mesenchymoma, malignant peripheral nerve sheath tumor, mesenchymal chondrosarcoma, myxofibrosarcoma, myxoid liposarcoma, myxoinflammatory fibroblastic sarcoma, neoplasms with perivascular epitheioid cell differentiation, osteosarcoma, parosteal osteosarcoma, neoplasm with perivascular epitheioid cell differentiation, periosteal osteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, PNET/extraskeletal Ewing tumor, rhabdomyosarcoma, round cell liposarcoma, small cell osteosarcoma, solitary fibrous tumor, synovial sarcoma, or telangiectatic osteosarcoma.

Exemplary carcinoma cell samples may include, but are not limited to, cell samples obtained from an anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

The cancerous cell sample may comprise cells obtained from a hematologic malignancy. Hematologic malignancy may comprise a leukemia, a lymphoma, a myeloma, a non-Hodgkin's lymphoma, or a Hodgkin's lymphoma. The hematologic malignancy may be a T-cell based hematologic malignancy. The hematologic malignancy may be a B-cell based hematologic malignancy. Exemplary B-cell based hematologic malignancy may include, but are not limited to, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, a non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. Exemplary T-cell based hematologic malignancy may include, but are not limited to, peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma, angioimmunoblastic lymphoma, cutaneous T-cell lymphoma, adult T-cell leukemia/lymphoma (ATLL), blastic NK-cell lymphoma, enteropathy-type T-cell lymphoma, hematosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma, nasal NK/T-cell lymphomas, or treatment-related T-cell lymphomas.

A cell sample described herein may comprise a tumor cell line sample. Exemplary tumor cell line sample may include, but are not limited to, cell samples from tumor cell lines such as 600MPE, AU565, BT-20, BT-474, BT-483, BT-549, Evsa-T, Hs578T, MCF-7, MDA-MB-231, SkBr3, T-47D, HeLa, DU145, PC3, LNCaP, A549, H1299, NCI-H460, A2780, SKOV-3/Luc, Neuro2a, RKO, RKO-AS45-1, HT-29, SW1417, SW948, DLD-1, SW480, Capan-1, MC/9, B72.3, B25.2, B6.2, B38.1, DMS 153, SU.86.86, SNU-182, SNU-423, SNU-449, SNU-475, SNU-387, Hs 817.T, LMH, LMH/2A, SNU-398, PLHC-1, HepG2/SF, OCI-Ly1, OCI-Ly2, OCI-Ly3, OCI-Ly4, OCI-Ly6, OCI-Ly7, OCI-Ly10, OCI-Ly18, OCI-Ly19, U2932, DB, HBL-1, RIVA, SUDHL2, TMD8, MEC1, MEC2, 8E5, CCRF-CEM, MOLT-3, TALL-104, AML-193, THP-1, BDCM, HL-60, Jurkat, RPMI 8226, MOLT-4, RS4, K-562, KASUMI-1, Daudi, GA-10, Raji, JeKo-1, NK-92, and Mino.

A cell sample may comprise cells obtained from a biopsy sample, necropsy sample, or autopsy sample.

The cell samples (such as a biopsy sample) may be obtained from an individual by any suitable means of obtaining the sample using well-known and routine clinical methods. Procedures for obtaining tissue samples from an individual are well known. For example, procedures for drawing and processing tissue sample such as from a needle aspiration biopsy are well-known and may be employed to obtain a sample for use in the methods provided. Typically, for collection of such a tissue sample, a thin hollow needle is inserted into a mass such as a tumor mass for sampling of cells that, after being stained, will be examined under a microscope.

A cell may be a live cell. A cell may be a eukaryotic cell. A cell may be a yeast cell. A cell may be a plant cell. A cell may be obtained from an agricultural plant.

EXAMPLES

These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Example 1 Genome Editing Complexes and Gene Repressors

This example describes genome editing complexes and gene repressors. A Ralstonia-derived modular nucleic acid binding domain (RNBD) is engineered by encoding for a plurality of repeat units, wherein each repeat unit is selected from any combination of SEQ ID NO: 168-SEQ ID NO: 263 or SEQ ID NO: 336-SEQ ID NO: 356. RNBDs are engineered to have an N-terminus as set forth in SEQ ID NO: 264 of SEQ ID NO: 303 and a C-terminus as set forth in SEQ ID NO: 266. The RNBD is engineered to also include a half repeat as set forth in SEQ ID NO: 265, prior to the C-terminus of SEQ ID NO: 266.

Genome Editing. The RNBD is linked to a nuclease, such as Fold or any one of SEQ ID NO: 1-SEQ ID NO: 81 (nucleic acid Sequences of SEQ ID NO: 82-SEQ ID NO: 162).

Gene Regulation. The RNBD is linked to an activator (e.g., VP16, VP64, p65, p300 catalytic domain, TET1 catalytic domain, TDG, Ldb1 self-associated domain, SAM activator (VP64, p65, HSF1), or VPR (VP64, p65, Rta) or a repressor (e.g., KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, or MeCP2).

Example 2 Mixed DNA Binding Domains

This example illustrates mixed DNA binding domains fused to nucleases to form genome editing complexes or fused to regulation domains to form gene activators or repressors. A Ralstonia-derived modular nucleic acid binding domain (RNBD) is engineered by encoding for a plurality of repeat units, wherein each repeat unit is selected from any combination of SEQ ID NO: 168-SEQ ID NO: 263 or SEQ ID NO: 336-SEQ ID NO: 356. RNBDs are engineered with an N-terminus as set forth in SEQ ID NO: 301 (Xanthomonas) or SEQ ID NO: 304 (Legionella). RNBDs are engineered with a C-terminus as set forth in SEQ ID NO: 298 (Xanthomonas) or SEQ ID NO: 306 (Legionella).

Genome Editing. The RNBD is linked to a nuclease, such as Fold or any one of SEQ ID NO: 1-SEQ ID NO: 81 (nucleic acid Sequences of SEQ ID NO: 82-SEQ ID NO: 162).

Gene Regulation. The RNBD is linked to an activator (e.g., VP16, VP64, p65, p300 catalytic domain, TET1 catalytic domain, TDG, Ldb1 self-associated domain, SAM activator (VP64, p65, HSF1), or VPR (VP64, p65, Rta) or a repressor (e.g., KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, or MeCP2).

Example 3 Genome Editing with an RNBD Fused to a Nuclease

This example illustrates genome editing with an RNBD fused to a nuclease. A first modular Ralstonia nucleic acid binding domain (RNBD) described herein, is fused to a cleavage half domain, such as an nuclease and a second modular Ralstonia DNA binding domain (RNBD) described herein, is fused to another cleavage half domain. The nucleic acid binding domains are fused to the nuclease, optionally, via a naturally occurring linker, a variant or truncation of a naturally occurring linker, or a synthetic linker. The first RNBD-nuclease complex recognizes a target nucleic acid sequence on the top strand of double stranded DNA and binds said region of the double stranded DNA and the second RNBD-nuclease complex recognizes a target nucleic acid sequence on the bottom strand of double stranded DNA and binds said region of the double stranded DNA. The 3′ end of the target nucleic acid sequence on the top strand and the 3′ end of the target nucleic acid sequence on the bottom strand are spaced 2 to 50 base pairs apart, referred to herein as the “spacer region.” Gene editing is carried out by dimerization of the two cleavage half domains in the spacer region followed by cleaving of the DNA phosphodiester bonds. Gene editing allows for the insertion of a sequence or deletion of a sequence.

Direct Administration to Introduce a Gene

The genome editing complex is administered directly to a subject in need thereof and is taken up by a cell. The subject has a disease. The DNA binding domain of the genome editing complex binds a region of DNA in a target cell and the cleavage domain induces a double strand break in the DNA of the target cell to introduce a gene. The introduced gene is a mutated gene or a functional gene.

Factor IX. The genome editing complex with a cleavage domain introduces a double strand break into the albumin gene locus (e.g., into intron 1) concomitant with delivery to the cell of an ectopic nucleic acid bearing a cDNA of the factor IX gene. The double strand break leads to the integration of the ectopic nucleic acid into intron 1 of the albumin gene; the factor IX protein is secreted by the cell into the circulation. The target cell is a hepatocyte and the subject in need thereof has Hemophilia B.

Ex Vivo Engineering of a Cell to Introduce a Gene

The genome editing complex is transfected into cells ex vivo along with an ectopic nucleic acid bearing a gene. Upon transfection of cells ex vivo, the DNA binding domain of the genome editing complex binds a region of DNA in a target cell and the cleavage domain induces a double strand break in the DNA of the target cell to introduce an ectopically provided gene (also provided to the cell) into the region cleaved by the genome editing complex. The resulting engineered cells with modified DNA are administered to a subject in need thereof. The subject has a disease.

CAR. The genome editing complex with a cleavage domain introduces a chimeric antigen receptor (CAR) by editing the DNA of a target cell. The target cell is a T cell and the subject has cancer, such as a blood cancer. Upon administration of the engineered cells to a subject, the engineered CAR T cells effectively eliminate cancer in the subject.

Direct Administration to Partially or Completely Knock Out a Gene

The genome editing complex is administered directly to a subject in need thereof and is taken up by a cell. The subject has a disease. The DNA binding domain of the genome editing complex binds a region of DNA in a target cell and the cleavage domain induces a double strand break in the DNA of the target cell to partially or completely knock out a gene.

TTR. The genome editing complex with a cleavage domain partially or completely knocks out the transthyretin (TTR) gene by editing the DNA of a target cell. The target cell is a liver cell and the subject in need thereof has transthyretin amyloidosis (ATTR).

SERPINA1. The genome editing complex with a cleavage domain partially or completely knocks out the SERPINA1 gene by editing the DNA of a target cell. The target cell is a liver cell and the subject in need thereof has alpha-1 antitrypsin deficiency (dA1AT def).

Ex Vivo Engineering of a Cell to Partially or Completely Knock Out a Gene or a Gene Regulatory Region

The genome editing complex is transfected in cells ex vivo. Upon transfection of cells ex vivo, the DNA binding domain of the genome editing complex binds a region of DNA in a target cell and the cleavage domain induces a double strand break in the DNA of the target cell to partially or completely knock out a gene or a gene regulatory region. The subject has a disease.

BCL11A Enhancer. The genome editing complex with a cleavage domain partially or completely knocks out the BCL11A erythroid enhancer by editing the DNA of a target cell. The target cell is an HPSC and the subject in need thereof has b-thalassemia or sickle cell disease.

CCR5. The genome editing complex with a cleavage domain partially or completely knocks the CCR5 gene by editing the DNA of a target cell, thereby allowing for introduction of a mutated version of CCR5. Target cells, in which mutated versions of CCR5 are introduced via the action of the genome editing complex, are not infected by HIV via the modified CCR5 receptor. The target cell is a T cell or a hematopoietic stem cell (HPSC) and the subject has HIV.

Upon administration of the genome editing complex directly to a subject or upon administration of an engineered cell with DNA that has been modified with the genome editing complex, the disease symptoms are eliminated or reduced.

Example 4 TALE Protein with N-Terminus Fragment

A DNA binding protein engineered to have a shortened N-terminus derived from a TALE protein was generated. U.S. Pat. No. 8,586,526 shows that while the N-terminus region (referred to as N-cap) from a TALE protein can be shortened by deleting amino acids at the N-terminus, deleting amino acids beyond amino acid position N+134 decreased DNA binding affinity, with the decrease in DNA binding apparent even with deletion of amino acids beyond amino acid position N+137. U.S. Pat. No. 8,586,526 concluded that amino acid sequence from N+1 through N+137 are required for binding to DNA while the first 152 amino acids of the N-cap sequence are dispensable.

However, it has been discovered that further deleting amino acids till position N+116 surprising leads to recovery of DNA binding. Even shorter N-terminus regions such as a fragment having deletion till position N+111 also retains DNA binding activity. Deleting amino acids till position N+106 significantly decreases DNA binding. Further deletion of the N-terminus region, such as, deleting amino acids till position N+101 does not lead to recovery of DNA binding. See FIG. 2.

TALEN monomers recognizing 5′-TTTCTGTCACCAATCCT-3′ and 5′-TCCCCTCCACCCCACAGT-3′ in the human AAVS1 locus were engineered to harbor N-terminus regions that included deletions encompassing residues N137-116, N137-111, N137-106 and N137-101. While these residues are numbered with reference to the N+137 construct in U.S. Pat. No. 8,586,526, N137-116 refers to deletion of amino acids starting at the N-terminus of the N-cap sequence (N+228) and extending through amino acid residue 116 such that the resulting fragment retains amino acids residues from position N+115 to position N+1, and so on. The amino acid sequence of the N-terminal truncation del_N137-116 is set forth in SEQ ID NO:321. The amino acid sequence of the N-terminal truncation del_N137-111 is set forth in SEQ ID NO:447.

NK562 cells were transfected with 2 μg plasmid DNA for each TALEN monomer using an AMAXA™ Nucleofector™ 96-well Shuttle™ system as per the manufacturer's recommendations. Full length TALEN monomers were included (“AAVS1 control”), together with N137-116/full length and full length/N137-116 heterodimers. Cells were cold shocked at 30° C. and genomic DNA was harvested at 72 h using QuickExtract™ (Lucigen). Indel rates were determined by amplicon sequencing. The TALE repeats present in the TALE monomers have the sequence LTPDQVVAIAS(RVD)GGKQALETVQRLLPVLCQDHG, with a RVD selected based on the target sequence.

FIG. 2 represents DNA binding activity assayed by measuring nuclease activity of FokI fused to C-terminus of the polypeptides. AAVS1 control data set correspond to TALENS using the standard full-length N-terminus (N+288 to N+1). N-terminal truncation del_N137-116 (N-terminus extending from N+115 to N+1) showed higher activity than standard full-length N-terminus (N+288 to N+1). N-terminal truncation del_N137-111 (N-terminus extending from N+110 to N+1) was also active. Further truncation del_N137-106 (N-terminus extending from N+105 to N+1) significantly decreased DNA binding. Further deletion of the N-terminus region del_N137-101 (N-terminus extending from N+100 to N+1) did not lead to recovery of DNA binding. Thus, a fragment of the N-terminus of a TALE protein extending from N+115 to N+1 shows full activity. Mock/GFP is a negative control. The AAVS1/del_N137-116 data shows that an N1-115 TALEN monomer can be combined with a monomer comprising full-length N-terminus region of a TALE protein.

While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. 

What is claimed is:
 1. A polypeptide comprising: a modular nucleic acid binding domain comprising a potency for a target site greater than 65% and a specificity ratio for the target site of at least 50:1; and a functional domain, wherein the functional domain is heterologous to the nuclear acid binding domain; wherein: the modular nucleic acid binding domain comprises a plurality of repeat units; at least one repeat unit of the plurality of repeat units comprises a binding region configured to bind to a target nucleic acid base within the target site; the potency comprises indel percentage at the target site, and wherein the specificity ratio comprises indel percentage at the target site over indel percentage at a top-ranked off-target site of the polypeptide.
 2. The polypeptide of claim 2, wherein the at least one repeat unit comprises a sequence of A₁₋₁₁X₁X₂B₁₄₋₃₅, wherein: each amino acid residue of A₁₋₁₁ comprises any amino acid residue; X₁X₂ comprises the binding region; each amino acid residue of B₁₄₋₃₅ comprises any amino acid; and a first repeat unit of the plurality of repeat units comprises at least one residue in A₁₋₁₁, B₁₄₋₃₅, or a combination thereof that differs from a corresponding residue in a second repeat unit of the plurality of repeat units.
 3. A polypeptide comprising a modular nucleic acid binding domain and a functional domain, wherein: the modular nucleic acid binding domain comprises a plurality of repeat units; at least one repeat unit of the plurality comprises a sequence of A₁₋₁₁ X₁X₂B₁₄₋₃₅; each amino acid residue of A₁₋₁₁ comprises any amino acid residue; X₁X₂ comprises a binding region configured to bind to a target nucleic acid base within a target site; each amino acid residue of B₁₄₋₃₅ comprises any amino acid; and a first repeat unit of the plurality of repeat units comprises at least one residue in A₁₋₁₁, B₁₄₋₃₅, or a combination thereof that differs from a corresponding residue in a second repeat unit of the plurality of repeat units.
 4. The polypeptide of any one of claims 1-3, wherein the binding region comprises an amino acid residue at position 13 or an amino acid residue at position 12 and the amino acid residue at position
 13. 5. The polypeptide of claim 4, wherein the amino acid residue at position 13 binds to the target nucleic acid base.
 6. The polypeptide of any one of claims 4-5, wherein the amino acid residue at position 12 stabilizes the configuration of the binding region.
 7. The polypeptide of any one of claims 3-6, wherein the modular nucleic acid binding domain further comprises a potency for the target site greater than 65% and a specificity ratio for the target site of at least 50:1, wherein the potency comprises indel percentage at the target site and the specificity ratio comprises indel percentage at the target site over indel percentage at a top-ranked off-target site of the polypeptide.
 8. The polypeptide of any one of claim 1, 2, or 4-7, wherein the indel percentage is measured by deep sequencing.
 9. The polypeptide of any one of claims 1-8, wherein the modular nucleic acid binding domain further comprises one or more properties selected from the following: (b) binds the target site, wherein the target site comprises a 5′ guanine; (c) comprises from 7 repeat units to 25 repeat units; (d) upon binding to the target site, the modular nucleic acid binding domain is separated from a second modular nucleic acid binding domain bound to a second target site by from 2 to 50 base pairs.
 10. The polypeptide of any one of claims 1-9, wherein the modular nucleic acid binding domain comprises a Ralstonia repeat unit.
 11. The polypeptide of claim 10, wherein the Ralstonia repeat unit is a Ralstonia solanacearum repeat unit.
 12. The polypeptide of any one of claims 2-11, wherein the B₁₄₋₃₅ of at least one repeat unit of the plurality of repeat units has at least 92% sequence identity to GGKQALEAVRAQLLDLRAAPYG (SEQ ID NO: 280).
 13. The polypeptide of any one of claims 1-12, wherein the binding region comprises HD binding to cytosine, NG binding to thymidine, NK binding to guanine, SI binding to adenosine, RS binding to adenosine, HN binding to guanine, or NT binds to adenosine.
 14. The polypeptide of any one of claims 1-13, wherein the at least one repeat unit comprises any one of SEQ ID NO: 267-SEQ ID NO:
 279. 15. The polypeptide of any one of claims 1-14, wherein the at least one repeat unit comprises at least 80% sequence identity with any one of SEQ ID NO: 168-SEQ ID NO:
 263. 16. The polypeptide of any one of claims 1-15, wherein the at least one repeat unit comprises at least 80% sequence identity with SEQ ID NO: 209, SEQ ID NO: 197, SEQ ID NO: 233, SEQ ID NO: 253, SEQ ID NO: 203, or SEQ ID NO:
 218. 17. The polypeptide of any one of claims 1-16, wherein the at least one repeat unit comprises any one of SEQ ID NO: 168-SEQ ID NO:
 263. 18. The polypeptide of any one of claims 1-17, wherein the at least one repeat unit comprises SEQ ID NO: 209, SEQ ID NO: 197, SEQ ID NO: 233, SEQ ID NO: 253, SEQ ID NO: 203, or SEQ ID NO:
 218. 19. The polypeptide of any one of claims 1-18, wherein the target nucleic acid base is cytosine, guanine, thymidine, adenosine, uracil or a combination thereof.
 20. The polypeptide of any one of claims 1-19, wherein the target site is a nucleic acid sequence within a PDCD1 gene, a CTLA4 gene, a LAG3 gene, a TET2 gene, a BTLA gene, a HAVCR2 gene, a CCR5 gene, a CXCR4 gene, a TRA gene, a TRB gene, a B2M gene, an albumin gene, a HBB gene, a HBA1 gene, a TTR gene, a NR3C1 gene, a CD52 gene, an erythroid specific enhancer of the BCL11A gene, a CBLB gene, a TGFBR1 gene, a SERPINA1 gene, a HBV genomic DNA in infected cells, a CEP290 gene, a DMD gene, a CFTR gene, an IL2RG gene, or a combination thereof.
 21. The polypeptide of any one of claims 1-20, wherein a nucleic acid sequence encoding a chimeric antigen receptor (CAR), alpha-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), or Factor 9 (F9), is inserted at the target site.
 22. The polypeptide of any one of claims 1-21, wherein the modular nucleic acid binding domain comprises an N-terminus amino acid sequence, a C-terminus amino acid sequence, or a combination thereof.
 23. The polypeptide of claim 22, wherein the N-terminus amino acid sequence is from Xanthomonas spp., Legionella quateirensis, or Ralstonia solanacearum.
 24. The polypeptide of any one of claims 22-23, wherein the N-terminus amino acid sequence comprises at least 80% sequence identity to SEQ ID NO: 264, SEQ ID NO: 300, SEQ ID NO: 335, SEQ ID NO: 303, SEQ ID NO: 301, SEQ ID NO: 304, or SEQ ID NO: 320, SEQ ID NO: 321, or SEQ ID NO:
 322. 25. The polypeptide of any one of claims 22-24, wherein the N-terminus amino acid sequence comprises SEQ ID NO: 264, SEQ ID NO: 300, SEQ ID NO: 335, SEQ ID NO: 303, SEQ ID NO: 301, SEQ ID NO: 304, or SEQ ID NO: 320, SEQ ID NO: 321, or SEQ ID NO:
 322. 26. The polypeptide of any one of claims 22-25, wherein the C-terminus amino acid sequence is from Xanthomonas spp., Legionella quateirensis, or Ralstonia solanacearum.
 27. The polypeptide of any one of claims 22-25, wherein the C-terminus amino acid sequence comprises at least 80% sequence identity to SEQ ID NO: 266, SEQ ID NO: 298, or SEQ ID NO:
 306. 28. The polypeptide of any one of claims 22-26, wherein the C-terminus amino acid sequence comprises SEQ ID NO: 266, SEQ ID NO: 298, or SEQ ID NO:
 306. 29. The polypeptide of any one of claims 22-28, wherein the C-terminus amino acid sequence serves as a linker between the modular nucleic acid binding domain and the cleavage domain.
 30. The polypeptide of any one of claims 1-29, wherein the modular nucleic acid binding domain comprises a half repeat.
 31. The polypeptide of claim 30, wherein the half repeat comprises at least 80% sequence identity to SEQ ID NO: 265, SEQ ID NO: 327-SEQ ID NO: 329, or SEQ ID NO:
 290. 32. The polypeptide of any one of claims 30-31, wherein the half repeat comprises SEQ ID NO: 265, SEQ ID NO: 327-SEQ ID NO: 329, or SEQ ID NO:
 290. 33. The polypeptide of any one of claims 1-32, wherein the functional domain is a cleavage domain or a repression domain.
 34. The polypeptide of claim 33, wherein the cleavage domain comprises at least 33.3% divergence from SEQ ID NO: 163 and is immunologically orthogonal to SEQ ID NO:
 163. 35. The polypeptide of any one of claims 33-34, comprising one or more of the following characteristics: (a) induces greater than 1% indels at a target site; (b) the cleavage domain comprises a molecular weight of less than 23 kDa; (c) the cleavage domain comprises less than 196 amino acids; (d) capable of cleaving across a spacer region greater than 24 base pairs.
 36. The polypeptide of any one of claims 33-35, wherein the polypeptide induces greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% indels at the target site.
 37. The polypeptide of any one of claims 33-36, wherein the cleavage domain comprises at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% divergence from SEQ ID NO:
 163. 38. The polypeptide of any one of claims 33-37, wherein the cleavage domain comprises a sequence selected from SEQ ID NO: 316-SEQ ID NO:
 319. 39. The polypeptide of any one of claims 33-38, wherein the cleavage domain comprises a nucleic acid sequence encoding for a sequence having at least 80% sequence identity with SEQ ID NO: 1-SEQ ID NO:
 81. 40. The polypeptide of any one of claims 33-38, wherein the cleavage domain comprises a nucleic acid sequence encoding for a sequence selected from SEQ ID NO: 1-SEQ ID NO:
 81. 41. The polypeptide of any one of claims 33-40, wherein the nucleic acid sequence comprises at least 80% sequence identity with SEQ ID NO: 82-SEQ ID NO:
 162. 42. The polypeptide of any one of claims 33-41, wherein the nucleotide sequence encoding for the sequence comprises any one of SEQ ID NO: 82-SEQ ID NO:
 162. 43. The polypeptide of claim 33, wherein the repression domain comprises KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, or MeCP2.
 44. The polypeptide of any one of claims 1-43, wherein the at least one repeat unit comprises 1-20 additional amino acid residues at the C-terminus.
 45. The polypeptide of any one of claims 1-44, wherein the at least repeat unit of the plurality of repeat units is separated from a neighboring repeat unit by a linker.
 46. The polypeptide of claim 45, wherein the linker comprises a recognition site.
 47. The polypeptide of claim 46, wherein the recognition site is for a small molecule, a protease, or a kinase.
 48. The polypeptide of claim 47, wherein the recognition site serves as a localization signal.
 49. The polypeptide of any one of claims 1-48, wherein the plurality of repeat units comprises 3 to 60 repeat units.
 50. The polypeptide of any one of claims 1-49, wherein a repeat unit of the plurality of repeat units recognizes a target nucleic acid base and wherein the plurality of repeat units has one or more of the following characteristics: (a) at least one repeat unit comprising greater than 39 amino acid residues; (b) at least one repeat unit comprising greater than 35 amino acid residues derived from the genus of Ralstonia; (c) at least one repeat unit comprising less than 32 amino acid residues; and (d) each repeat unit of the plurality of repeat units is separated from a neighboring repeat unit by a linker comprising a recognition site.
 51. The polypeptide of claim 50, wherein the at least one repeat unit comprises an amino acid selected from glycine, alanine, threonine or histidine at a position after an amino acid residue at position
 35. 52. The polypeptide any one of claims 50-51, wherein the at least one repeat unit comprises an amino acid selected from glycine, alanine, threonine or histidine at a position after an amino acid residue at position
 39. 53. A method of genome editing, the method comprising: administering the polypeptide of any one of claim 1-42 or 44-52 and inducing a double stranded break.
 54. A method of gene repression, the method comprising administering the polypeptide of any one of claim 1-33 or 43-52 and repressing gene expression.
 55. A non-naturally occurring DNA-binding polypeptide comprising from N-terminus to C-terminus: an N-terminus region comprises at least residues N+110 to N+1 of a TALE protein, wherein the N-terminus region does not include residues N+288 to N+116 of the TALE protein; a plurality of TALE-repeat units, the TALE repeat units comprising a repeat variable di-residue (RVD); and a C-terminus region of a TALE protein.
 56. The DNA binding polypeptide of claim 55, wherein the N-terminus region comprises residues N+1 up to N+115 of the TALE protein.
 57. The DNA binding polypeptide of claim 55, wherein the N-terminus region comprises residues N+1 up to N+110 of the TALE protein.
 58. The DNA binding polypeptide of any one of claims 55-57, wherein the C-terminus region comprises residues C+1 to C+63 of the TALE protein.
 59. The DNA binding polypeptide of any one of claims 55-58, wherein the N-terminus region consists of residues N+1 to N+115 of the TALE protein.
 60. The DNA binding polypeptide of any one of claims 55-59, wherein the TALE repeat units are ordered from N-terminus to C-terminus to specifically bind to a target nucleic acid in genomic DNA.
 61. The DNA binding polypeptide of any one of claims 55-60, wherein a heterologous functional domain is conjugated to the N-terminus and/or C-terminus.
 62. The DNA binding polypeptide of claim 61, wherein the functional domain comprises an enzyme, a transcriptional activator, a transcriptional repressor, or a DNA nucleotide modifier.
 63. The DNA binding polypeptide of claim 62, wherein the enzyme is a nuclease, a DNA modifying protein, or a chromatin modifying protein.
 64. The DNA binding polypeptide of claim 63, wherein the nuclease is a cleavage domain or a half-cleavage domain.
 65. The DNA binding polypeptide of claim 64, wherein the cleavage domain or half-cleavage domain comprises a type IIS restriction enzyme.
 66. The DNA binding polypeptide of claim 65, wherein the type IIS restriction enzyme comprises FokI or Bfil.
 67. The DNA binding polypeptide of claim 63, wherein the chromatin modifying protein is lysine-specific histone demethylase 1 (LSD1).
 68. The DNA binding polypeptide of claim 62, wherein the transcriptional activator comprises VP16, VP64, p65, p300 catalytic domain, TET1 catalytic domain, TDG, Ldb1 self-associated domain, SAM activator (VP64, p65, HSF1), or VPR (VP64, p65, Rta).
 69. The DNA binding polypeptide of claim 62, wherein the transcriptional repressor comprises KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, or MeCP2.
 70. The DNA binding polypeptide claim 62, wherein the DNA nucleotide modifier is adenosine deaminase.
 71. The DNA binding polypeptide of any of claims 60-70, wherein the target nucleic acid is within a PDCD1 gene, a CTLA4 gene, a LAG3 gene, a TET2 gene, a ETLA gene, a HA VCR2 gene, a CCR5 gene, a CXCR4 gene, a TRA gene, a TRE gene, a E2M gene, an albumin gene, a HEE gene, a HEAl gene, a TTR gene, a NR3Cl gene, a CD52 gene, an erythroid specific enhancer of the ECLllA gene, a CELE gene, a TGFERl gene, a SERPINAl gene, a HEV genomic DNA in infected cells, a CEP290 gene, a DMD gene, a CFTR gene, or an IL2RG gene.
 72. The DNA binding polypeptide of any of claims 60-71, wherein the heterologous functional domain comprises a fluorophore or a detectable tag.
 73. A nucleic acid encoding the polypeptide of any of claims 55-73.
 74. The nucleic acid of claim 73, wherein the nucleic acid is operably linked to a promoter sequence that confers expression of the polypeptide.
 75. The nucleic acid of claim 73 or 74, wherein the sequence of the nucleic acid is codon optimized for expression of the polypeptide in a human cell.
 76. The nucleic acid of any one of claims 73-75, wherein the nucleic acid is a deoxyribonucleic acid (DNA).
 77. The nucleic acid of any one of claims 73-75, wherein the nucleic acid is a ribonucleic acid (RNA).
 78. A vector comprising the nucleic acid of any of claims 73-76.
 79. The vector of claim 78, wherein the vector is a viral vector.
 80. A host cell comprising the nucleic acid of any of claims 73-77 or the vector of claim 78 or
 79. 81. A host cell that expresses the polypeptide of any of claims 55-72.
 82. A pharmaceutical polypeptide comprising the polypeptide of any of claims 55-72 and a pharmaceutically acceptable excipient.
 83. A pharmaceutical polypeptide comprising the nucleic acid of any of claims 73-77 or the vector of claim 78 or 79 and a pharmaceutically acceptable excipient.
 84. A method of modulating expression of an endogenous gene in a cell, the method comprising: introducing into the cell the polypeptide of claim 61, wherein the DNA binding polypeptide binds to a target nucleic acid sequence present in the endogenous gene and the heterologous functional domain modulates expression of the endogenous gene.
 85. The method of claim 84, wherein the polypeptide is introduced as a nucleic acid encoding the polypeptide.
 86. The method of claim 85, wherein the nucleic acid is a deoxyribonucleic acid (DNA).
 87. The method of claim 85, wherein the nucleic acid is a ribonucleic acid (RNA).
 88. The method of any of claims 85-87, wherein the sequence of the nucleic acid is codon optimized for expression in a human cell.
 89. The method of any of claims 84-88, wherein the functional domain is a transcriptional activator and the target nucleic acid sequence is present in an expression control region of the gene, wherein the polypeptide increases expression of the gene.
 90. The method of claim 89, wherein the transcriptional activator comprises VP16, VP64, p65, p300 catalytic domain, TET1 catalytic domain, TDG, Ldb1 self-associated domain, SAM activator (VP64, p65, HSF1), or VPR (VP64, p65, Rta).
 91. The method of any of claims 84-88, wherein the functional domain is a transcriptional repressor and the target nucleic acid sequence is present in an expression control region of the gene, wherein the polypeptide decreases expression of the gene.
 92. The method of claim 91, wherein the transcriptional repressor comprises KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, or MeCP2.
 93. The method of any of claims 84-92, wherein the gene is a PDCD1 gene, a CTLA4 gene, a LAG3 gene, a TET2 gene, a ETLA gene, a HA VCR2 gene, a CCR5 gene, a CXCR4 gene, a TRA gene, a TRE gene, a E2M gene, an albumin gene, a HEE gene, a HEAl gene, a TTR gene, a NR3Cl gene, a CD52 gene, an erythroid specific enhancer of the ECLllA gene, a CELE gene, a TGFERl gene, a SERPINAl gene, a HEV genomic DNA in infected cells, a CEP290 gene, a DMD gene, a CFTR gene, or an IL2RG gene.
 94. The method of any of claims 91-93, wherein the expression control region of the gene comprises a promoter region of the gene.
 95. The method of any of claims 84-88, wherein the functional domain is a nuclease comprising a cleavage domain or a half-cleavage domain and the endogenous gene is inactivated by cleavage.
 96. The method of claim 95, wherein inactivation occurs via non-homologous end joining (NHEJ).
 97. The method of claim 95 or 96, wherein the DNA binding polypeptide is a first polypeptide that binds to a first target nucleic acid sequence in the gene and comprises a half-cleavage domain and the method comprises introducing a second DNA binding polypeptide that binds to a second target nucleic acid sequence in the gene and comprises a half-cleavage domain.
 98. The method of claim 97, wherein the first target nucleic acid sequence and the second target sequence are spaced apart in the gene and the two half-cleavage domains mediate a cleavage of the gene sequence at a location in between the first and second target nucleic acid sequences, wherein the second DNA binding polypeptide comprises from N-terminus to C-terminus: an N-terminus region comprising residues N+1 to up to N+115 of a TALE protein or a full-length N-terminus region of a TALE protein; a plurality of TALE-repeat units, the TALE repeat units comprising a repeat variable di-residue (RVD); and a C-terminus region of a TALE protein.
 99. The method of claim 98, wherein the C-terminus region is a full length region of the TALE protein.
 100. The method of claim 98, wherein the C-terminus region is a fragment of the C-terminus region of the TALE protein.
 101. The method of claim 98, wherein the C-terminus region extends from C+1 to C+63 of the TALE protein.
 102. The method of any of claims 95-101, wherein the cleavage domain or the cleavage half domain comprises FokI or Bfil.
 103. The method of any of claims 95-101, wherein the cleavage domain comprises a meganuclease.
 104. The method of any of claims 95-103, wherein the gene is a PDCD1 gene, a CTLA4 gene, a LAG3 gene, a TET2 gene, a ETLA gene, a HA VCR2 gene, a CCR5 gene, a CXCR4 gene, a TRA gene, a TRE gene, a E2M gene, an albumin gene, a HEE gene, a HEAl gene, a TTR gene, a NR3Cl gene, a CD52 gene, an erythroid specific enhancer of the ECLllA gene, a CELE gene, a TGFERl gene, a SERPINAl gene, a HEV genomic DNA in infected cells, a CEP290 gene, a DMD gene, a CFTR gene, or an IL2RG gene. 